Farnesyltransferase inhibitors for treatment of laminopathies, cellular aging and atherosclerosis

ABSTRACT

Although it can be farnesylated, the mutant lamin A protein expressed in Hutchinson Gilford Progeria Syndrome (HGPS) cannot be defarnesylated because the characteristic mutation causes deletion of a cleavage site necessary for binding the protease ZMPSTE24 and effecting defarnesylation. The result is an aberrant farnesylated protein (called “progerin”) that alters normal lamin A function as a dominant negative, as well as assuming its own aberrant function through its association with the nuclear membrane. The retention of farnesylation, and potentially other abnormal properties of progerin and other abnormal lamin gene protein products, produces disease. Farnesyltransferase inhibitors (FTIs) (both direct effectors and indirect inhibitors) will inhibit the formation of progerin, cause a decrease in lamin A protein, and/or an increase prelamin A protein. Decreasing the amount of aberrant protein improves cellular effects caused by and progerin expression. Similarly, treatment with FTIs should improve disease status in progeria and other laminopathies. In addition, elements of atherosclerosis and aging in non-laminopathy individuals will improve after treatment with farnesyltransferase inhibitors.

CROSS-REFERENCE TO RELATED APPLICATIONS

This is a division of co-pending U.S. patent application Ser. No.11/828,117, filed Jul. 25, 2007, which is a continuation-in-part ofInternational Application No. PCT/US2006/002977, filed Jan. 27, 2006,which in turn claims the benefit of U.S. Provisional Application No.60/648,307, filed Jan. 28, 2005 and U.S. Provisional Application No.60/707,192, filed Aug. 9, 2005; and a continuation-in-part of U.S.Utility application Ser. No. 10/943,400, filed Sep. 17, 2004, whichissued as U.S. Pat. No. 7,297,942 on Nov. 20, 2007, which is acontinuation of International Application No. PCT/US2003/033058, filedOct. 17, 2003, which in turn claims the benefit of U.S. ProvisionalApplication No. 60/463,084, filed Apr. 14, 2003, and U.S. ProvisionalApplication No. 60/419,541, filed Oct. 18, 2002. Each of theseapplications is incorporated herein in their entirety.

FIELD

This disclosure relates to treatment of laminopathies, cellular agingand aging-related conditions, and more particularly to the use offarnesyltransferase inhibitors (FTIs) and other compounds andcompositions to treat such conditions. It also relates to methods ofidentifying agents useful in treating, for instance, laminopathies.

BACKGROUND

Hutchinson-Gilford Progeria Syndrome (HGPS) is a rare genetic diseasethat affects children in the first decade of life and causes aremarkable phenotype resembling many aspects of aging. Affected childrendevelop an extremely aged appearance, a lack of subcutaneous fat, growthretardation and severe atherosclerosis. Affected children die ofpremature atherosclerosis at an average age of 13 years. Progeria is adisease in which some, but not all, of its manifestations (in vivo andin vitro) represent a model of accelerated aging (reviewed in Sweeney &Weiss, Gerontology 38:139-52, 1992). Clinical features common toprogeria and normal aging include alopecia (although the pattern of hairloss differs), sclerodermatosis, atherosclerosis, lipofuscin deposition,nail dystrophy, hypermelanosis, decreased adipose tissue, andosteoporosis. Clinical differences include sequelae of maldevelopment inprogeria, with coxa valga, distal bone resorption, delayed dentition,facial disproportion, failure to thrive, and short stature. Features ofaging that are absent in progeria include neurosensory decline such asAlzheimer's disease, dementia, hearing loss, and presbyopia.

Recently, the gene responsible for HGPS was identified, and HGPS joineda group of syndromes—the laminopathies—all of which have an underlyingdefect in the lamin A/C gene (LMNA) (Eriksson et al., Nature423:293-298, 2003). LMNA codes for the lamin A and lamin C isoforms,which differ due to alternate splicing, as well as the Δ10 isoform foundin sperm. The lamins are a component of the nuclear lamina, a fibrousmatrix located at the interior of the nuclear membrane, responsible fornuclear integrity and organization. In addition, lamins are also presentin the nucleoplasm and may be involved in more complex spatialorganization of the nucleus. They play a role in a wide array of nuclearprocesses, including transcription, replication, chromatin organization,nuclear shape, cell division, and cell cycle functions (Gruenbaum etal., J Struct Biol 129:313-23, 2000; Gruenbaum et al., Nat. Rev. Mol.Cell Biol. 6:21-31, 2005). The LMNA gene is primarily expressed indifferentiated tissues in the fetus and adult and may be important inmaintaining the differentiated state (Rober et al., Development105:365-378, 1989). In fact, lamin A expression is down-regulated inmany tumors, perhaps as part of the loss of differentiation seen inthose tumors (Muller et al., Leukemia 8:940-945, 1994).

The pre-lamin A protein contains a CAAX box (SEQ ID NO: 31) at thecarboxy terminus, which is an invariant cysteine followed by twoaliphatic amino acids with the X denoting the terminal amino acid. TheCAAX box signals for isoprenylation, the addition of a 15-carbonfarnesyl isoprenoid lipid group to the cysteine by the enzymefarnesyltransferase (FTase) or a 20-carbon geranylgeranyl isoprenoidlipid by geranylgeranyltransferase I (GGTaseI) (Beck et al., J. CellBiol. 110:1489-1499, 1990). The final amino acid defines the specificityfor the addition of the isoprenyl group with methionine, serine,glutamine, or alanine signaling farnesylation and leucine signaling theaddition of a 20-carbon geranylgeranyl isoprenoid group catalyzed by thestructurally related enzyme GGTase I (Moores et al., J Biol Chem266:14603-14610, 1991; Cox & Der, Curr. Opin. Pharmacol. 2:388-393,2002). The native lamin A CAAX box (SEQ ID NO: 31) consists of CSIM(cysteine, serine, isoleucine, methionine) (SEQ ID NO: 32).Farnesylation, together with subsequent CAAX-signaled modifications,promote prelamin A association with the nuclear membrane (Hennekes &Nigg, J. Cell Sci. 107:1019-1029, 1994). Farnesylation is a permanentmodification; once a farnesyl group is added to a protein, it remainsattached to that residue for the life of the protein. Followingfarnesylation, the terminal three AAX amino acids are removed, and theC-terminal isoprenylated cysteine undergoes methyl esterification(Hennekes & Nigg, J. Cell Sci. 107:1019-1029, 1994). While both B-typelamins and lamin A are farnesylated and carboxymethylated, unique tolamin A is a second cleavage that occurs inside the nucleus causing theremoval of an additional 15 C-terminal amino acids from the matureprotein, including the farnesylated cysteine. Because farnesylation is apermanent posttranslational modification, proteolytic cleavage of thefarnesylated cysteine is necessary for full processing of the prelamin Aprotein to mature lamin A, and for its correct subcellular localizationand function. Thus, this final cleavage step and the resulting loss ofthe farnesyl anchor presumably releases prelamin A from the nuclearmembrane and allows it to be inserted into the nuclear lamina. In HGPS,although preprogerin can be farnesylated, its internal deletion of aminoacids 606-656 removes the endoprotease recognition site necessary forexecuting the final cleavage step. This final cleavage step appears tobe important for normal function as mutations in ZMPSTE24 cause a severeform of mandibuloacral dysplasia (MADB), one of the laminopathies whichis phenotypically similar to HGPS (Agarwal et al., Hum. Mol. Genet.12:1995-2001, 2003). ZMPSTE24 is the human homolog of yeast STE 24 andis responsible for the final cleavage of lamin A that removes the 15terminal amino acids (Pendas et al., Nat. Genet. 31:94-99, 2002).

Nearly all HGPS patients have the same silent mutation (G608G) creatingan abnormal splice donor site in exon 11 of the LMNA gene (Eriksson etal., Nature 423:293-298, 2003), which causes a 150 base pair mRNAdeletion in the lamin A transcript. The result of the mis-splicing is aprotein missing 50 amino acids near the C-terminus (henceforth called“preprogerin” prior to posttranslational processing and “progerin” afterpost-translational processing). The deleted region includes the proteincleavage site that normally removes the C-terminal 15 amino acids,including the farnesylated cysteine. The deleted region also containstwo potential cyclin-dependent kinase target serines (652 and 657) thatmay be involved in dissociation and reassociation of the nuclearmembrane at each cell division (Sinensky et al., J Cell Sci 107 (Pt1):61-7, 1994; Kilic et al., J Biol Chem 272(8):5298-304, 1997) and itmay affect molecular solubility (Hennekes & Nigg, J Cell Sci.107:1019-29, 1994).

SUMMARY OF THE DISCLOSURE

Although it can be farnesylated, the progerin protein cannot bedefarnesylated because the characteristic mutation causes deletion ofthe second cleavage site necessary for binding ZMPSTE24 and effectingdefarnesylation (Eriksson et al., Nature 423:293-298, 2003). The resultis a shortened, aberrant (abnormally) farnesylated protein capable ofaltering normal lamin A function as a dominant negative, as well asassuming its own aberrant function through its association with thenuclear membrane. Thus, the multisystem disease process and the varietyof genes that are affected in HGPS (Csoka et al., Aging Cell 3:235-243,2004) lie downstream of a protein defect that is central to basiccellular function. Other lamin defects also lead to disease processes(generally referred to as laminopathies) due to aberrant properties ofthe LMNA protein product.

The retention of farnesylation, and potentially other abnormalproperties of progerin and other abnormal lamin gene protein products,produces disease. Farnesyltransferase inhibitors (both direct effectorsand indirect inhibitors of farnesyltransferase) will preventfarnesylation, inhibit the formation of progerin, cause a decrease inlamin A protein and an increase in prelamin A and preprogerin proteins.Decreasing the amount of aberrant protein will improve disease status inProgeria and other laminopathies (in that the status will be movedtowards normal). In addition, altering lamin A using farnesyltransferaseinhibitors (both direct effectors and indirect inhibitors) will improvethose elements of other diseases and conditions, such as atherosclerosisand aging, in normal individuals that involve lamin A.

The foregoing and other features and advantages will become moreapparent from the following detailed description of several embodiments,which proceeds with reference to the accompanying figures.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows a schematic overview of normal lamin A processing. TheC-terminal CAAX box (SEQ ID NO: 31) of prelamin A undergoesfarnesylation, which allows localization of the partially processedprotein to the nuclear membrane. The AAX endopeptidase cleaves the lastthree amino acids from the farnesylated protein. Prelamin A thenundergoes methyl esterification at the C-terminus. The endoproteaseZMPSTE24 then cleaves the c-terminal 15 amino acids, which cleavagereleases a short peptide containing both the farnesylation and themethyl ester. This last proteolytic cleavage is prevented in progerin,as the cleavage site is lost in the 50 amino acid deletion. In addition,neither proteolytic cleavage can occur without prior farnesylation ofthe precursor protein.

FIG. 2 is a diagram showing the differential processing of lamin A exon11 in wild-type and the HGPS mutant. The silent mutation G608G leads toactivation of a cryptic splice site, which results in splicing-out of150 nucleotides corresponding to the 3′-end of Exon 11.

FIG. 3 shows drawings of prelamin A, lamin A, and progerin. The deletionin progerin removes amino acids 608-657 of the wildtype lamin A protein;this region includes both a phosphorylation site (indicated in thefigure by an asterisk) and a proteolytic cleavage site that is requiredfor maturation from the pre-form to the mature lamin A. The nuclearlocalization sequence (NLS) is unaffected by the mutation.

FIG. 4 illustrates nuclear morphology and protein localization in cellstransfected with plasmid DNA encoding green fluorescent protein(GFP)-tagged lamin A or GFP-tagged wild type or mutant progerinproteins. GFP-lamin A (panels A and B) or GFP-progerin (panel C)expression constructs were transiently transfected into the normalfibroblast line, GM08398, using FUGENE® transfection reagent (Roche).Seventy-two hours later the cells were fixed with 4% paraformaldehydeand visualized using a Zeiss Axiophot fluorescence microscope with aSensys CCD cameras and Applied Imaging digital imaging capture software.The actin cytoskeleton was stained with Rhodamine-phalloidin. Panel Ashows the same cell as in Panel B but with the focal plane bisecting thenucleus. In B and C the periphery of the nucleus is in focus to show thealtered localization of GFP-progerin in panel C.

FIG. 5 shows images of confocal microscopy of transfected cells. Normalfibroblasts were plated on coverslips, transfected, and fixed as in FIG.4. Localization of GFP-lamin A (panel A) and progerin (panel B) wasanalyzed using a Zeiss LSM 510 confocal microscope mounted on a ZeissAxiovert 100M inverted microscope. Three focal planes are shown-top,middle and bottom (from left to right). These images show the locationof the GFP-progerin aggregates as thicker regions at the nuclearperiphery (B). Fibroblasts expressing normal GFP-lamin A have GFP signalaround the nuclear periphery (A).

FIG. 6 illustrates immunocytochemistry using a lamin A-specific antibodyto detect localization of endogenous lamin A. Normal (A and B) and HGPS(C and D) fibroblasts were grown on coverslips, fixed with formaldehydeand probed with anti-lamin A antibodies (USBiological). Lamin Aaggregates were observed in HGPS fibroblasts. Images show two focalplanes, a nuclear cross-section (A and C) and a focal plane bisectingthe nucleus (B and D).

FIG. 7 illustrates the effect of a 72 hour exposure to 100 nM FTI(PD169451) on GFP-lamin A (A) and GFP-progerin (B) localization, proteinprocessing (C) and nuclear morphology (D and E). Confocal microscopy wasused to examine the effects of FTIs on transiently-expressed GFP-lamin A(A) and GFP-progerin (B) in normal fibroblasts. Three focal planes areshown—top, middle and bottom (from left to right). Exposure to 100 nMFTI for 72 hours caused about half of the endogenous lamin A to beunprocessed pre-lamin A in normal and HGPS fibroblasts (C). Exposure tothe FTI increased the percent of mock transfected cells and GFP-lamin Aexpressing cells with abnormal nuclear morphology (D). However, thepercentage of cells expressing GFP-progerin with an abnormal nuclearmorphology decreased by 33% when exposed to the FTI for 72 hours. Thenuclear morphology of HGPS fibroblasts showed a similar improvement inthe percent of abnormal nuclei when exposed to the FTI for 72 hours (E).

FIG. 8 shows the nuclear morphology of normal fibroblasts (GM08398)expressing GFP-lamin A (panel A), GFP-progerin (panel B), and GFP-laminA L647R (panel C). DAPI staining of nuclei is shown on the left and GFPsignal on the right. The GFP constructs were expressed from the pRevTREretroviral Tet-Off expression vector for three days by removingtetracycline from the growth medium followed by fixing the cells with 4%paraformaldehyde and mounting with ProLong Gold with DAPI.

FIG. 9 illustrates the effect of expressing the cleavage minus mutant(GFP-lamin A L647R) on nuclear morphology. GFP-lamin A, GFP-progerin,and GFP-lamin A L647R were expressed in the normal fibroblast line,GM08398, for three days followed by fixing the cells with 4%paraformaldehyde and mounting with ProLong Gold with DAPI. Abnormalnuclear morphology was assessed as nuclei with multiple blebs. Theelevated proportion of abnormal nuclei in the presence of tetracycline(+Tet) in the GFP-progerin and GFP-LA L647R is due to the leakyexpression of these proteins in the concentration of tetracycline usedto prevent expression.

FIG. 10 illustrates the effect of long-term exposure to low doses of FTI(PD169451) on the nuclear morphology of normal (GM08398 and HGMDFN090)and HGPS (AG11498, P01, HGADFN136 and HGADFN004) fibroblasts.Fibroblasts were maintained in medium with 0 nM, 10 nM or 100 nM FTI.Cultures were plated in chambered microscope slides 24 hours prior tofixing with 4% paraformaldehyde for nuclear morphology studies. **-Toofew cells to count. The HGADFN004 line failed to grow in 100 nM FTI tothe 14 day time point. All cell lines were passage 6 at the beginning ofthe analysis except for HGADFN004, which was at passage 12.

FIG. 11 illustrates the effect of the FTI, PD169451, on the nuclearmorphology of normal fibroblasts (GM08398) stably expressing GFP-laminA, GFP-progerin and GFP-LA(L647R) using a retroviral Tet-off system(Clontech). Fibroblasts were maintained in medium with 0 nM, 10 nM or100 nM FTI for 6 days.

Cultures were plated in chambered microscope slides 24 hours prior tofixing with 4% paraformaldehyde for nuclear morphology studies. Cellswere maintained in three predetermined tetracycline concentrations, 0 mMfor full expression (A), 0.05 mM for intermediate expression levels (B),and 8 mM for very low expression levels (C). Using a Tet-off systemallowed for modulation of expression levels with tetracyclineconcentration as demonstrated by immunoblot of cells from the aboveexperiment (D). When GFP-progerin and GFP-LA(L647R) mutant was stablyexpressed in normal fibroblasts at intermediate levels (0.05 mg/mltetracycline), 10 nM FTI substantially reduced the percent abnormalnuclei by 71% and 67%, respectively (p<0.0001) (B). Analysis of nuclearmorphology was performed in a double blinded fashion with at least 70cells analyzed per sample.

FIG. 12 illustrates the effect of FTI on the distribution of GFP signalin normal fibroblasts (GM08398) expressing GFP-lamin A (panels A, B andC), GFP-progerin (D, E and F) and GFP-LA(L647R) (G, H, and I) using aretroviral Tet-off system with various FTI concentrations. Fibroblastswere maintained in medium with 0 nM (A, D, G), 10 nM (B, E, and H) or100 nM (C, F and I) FTI for six days. Expression levels of the GFPfusions were maintained at levels similar to endogenous levels of laminA by the addition of 0.05 mg/ml tetracycline. Signals were visualizedwith a Zeiss Axiophot Fluorescent microscope and imaging was performedusing the Olympus DP70 Digital Camera System.

FIG. 13 includes the maps of three constructs discussed in Examples 5and 6. MAP 1 illustrates a representative cDNA construct expressingEGFP-labeled lamin A, with or without the 150-nucleotide deletion usedto mimic HGPS lamin A. The construct is expressed under the control of aCMV promoter, and also contains a myc epitope tag. MAP 2 illustrates arepresentative minigene construct expressing EGFP-labeled lamin A, withor without the 150-nucleotide deletion used to mimic HGPS lamin A. Theconstruct is expressed under the control of a CMV promoter, and alsocontains a myc epitope tag. MAP 3 illustrates a representativetet-inducible minigene construct expressing lamin A, with or without the150-nucleotide deletion used to mimic HGPS lamin A (Tet 75/77-lamin Aminigene constructs). One specific example of a Tet-inducible minigeneconstruct is Tet 75-lamin A, which contains the G608G mutation (GCG toGGT) causing partial aberrant splicing from Exon 11 to Exon 12 anddeleting 150 nucleotides (corresponding to 50 amino acid residues),thereby producing a progerin-like protein. Another is Tet 77-lamin A,which contains the wild type lamin A minigene.

FIG. 14 is a series of schematic drawings of lamin A and mutant lamin Aexpression constructs.

FIG. 15 is a Western blot illustrating expression of Progerin fromfibroblasts transfected with Progerin cDNA and minigene: M=Molecularweight marker; C1 Normal Fibroblast untransfected; GFP−P=NormalFibroblast transfected with complete Progerin cDNA construct;GFP−MG6=Normal Fibroblast transfected with MG6-minigene which shows thesplicing event.

FIG. 16 is a series of digital scans of nucleic acid gels, showingprocessing of lamin and progerin minigene constructs in human vascularcells. FIG. 16A shows results in EC; FIG. 16B shows coronary artery SMC;and FIG. 16C shows radial artery SMC. Each cell type expresses andprocesses progerin after minigene transcript transfection. Cells wereelectroporated and exposed to either Progerin minigene (MG) orLMNA-lamin A (L). Arrows indicate the 489 base pair band of interest inthe MG lane, produced by the Progeria gene which indicates a splicingevent occurring in VSMC and EC. Other bands indicate incomplete splicingof the minigene.

FIG. 17 shows population doubling times of cultured human dermalfibroblast lines. Solid lines indicate 4-parameter exponential growthcurve fit for HGPS (line through triangles) and Normal (line throughcircles). Passage doubling (PD) time at each passage is indicated byopen triangles (HGPS) and open circles (Normal). Filled diamondindicates a second HGPS cell line at later passage for comparison.Corresponding SAβ-gal staining is indicated below. Corresponding SAβ-galstaining is indicated below each letter.

FIG. 18: Cells were stained with Annexin V for apoptosis, PropidiumIodide (PI) for necrosis, and DAPI for DNA (all 20×). FIG. 18A:Non-apoptotic normal fibroblasts (passage 14) display round nuclei. FIG.18B: HGPS fibroblasts (passage 14) display apoptosis with underlyingnuclei. FIG. 18C: HGPS fibroblasts immortalized with hTERT catalyticsubunit of telomerase show no apoptosis (passage 35).

FIG. 19: Cells were plated on glass coverslips and stained for lamin A/C(upper and lower panels) and DAPI (lower panels). Images were takenunder 40× objective and analyzed for nuclear morphology. HGPS cells(B&D) exhibit blebbing of the nuclear envelope whereas Normal cells(A&C) do not.

FIG. 20: Normal (n=6) and HGPS (n=6) fibroblast cell lines were assessedfor MMP-3 secretion. MMP-3 secretion from HGPS fibroblasts wassignificantly (p=0.003) decreased over controls.

FIG. 21. Translation of the LMNA gene yields the prelamin A protein,which requires posttranslational processing for incorporation into thenuclear lamina. The prelamin protein contains a CAAX box (CSIM) (SEQ IDNO: 31) at the C-terminus signaling isoprenylation, in this caseaddition of a farnesyl group to the cysteine by the enzymefarnesyltransferase. Following farnesylation, the terminal three aminoacids (SIM) are cleaved, and the terminal farnesylated cysteineundergoes methyl esterification. A second cleavage step by the ZMPSTE24endoprotease then removes the terminal 15 amino acids, including thefarnesyl group. This final cleavage step is blocked in progeria.

FIG. 22. Subcellular localization and induction of nuclear blebbing byprenylation mutants of wild-type and progerin lamin A. Transienttransfection of HeLa cells with expression vectors encoding theindicated wild-type or progerin lamin A protein. (a) Normal wildtypelamin A-CSIM CAAX motif (SEQ ID NO: 31); (b) wild-type lamin A with theCSIM sequence (SEQ ID NO: 32) mutated to SSIM (SEQ ID NO: 33) to preventfarnesylation (because serine (S) cannot be farnesylated), relocalizingall lamin A into nucleoplasmic aggregates; (c) wild-type lamin A withthe CSIM sequence (SEQ ID NO: 32) mutated to CSIL (SEQ ID NO: 34),predicted to be geranylgeranylated by GGTase I, resulting in a mix ofnucleoplasmic and nuclear membrane localization; (d) the progerin-CSIMconstruct recreates the hallmark nuclear blebbing seen in HGPS; (e) whenthe CSIM (SEQ ID NO: 32) of the progerin-CSIM is mutated to SSIM (SEQ IDNO: 33) (progerin-SSIM), blebbing is prevented, and progerin accumulatesin nucleoplasmic aggregates; (f) progerin-CSIL causes blebbingindistinguishable from that of authentic progerin-CSIM. Cells werevisualized 48 hours after transfection and photomicrographs were takenat 60× magnification.

FIG. 23: FTI treatment prevents nuclear blebbing caused by farnesylated,but not geranylgeranylated, progerin. HeLa cells were transientlytransfected with expression vectors encoding either the progerin-CSIM,progerin-SSIM or progerin-CSIL constructs, and were treated with either0 or 2 μM of the FTI lonafarnib/SCH66336. (a) The progerin-CSIMconstruct recreates the hallmark nuclear blebbing seen in HGPS, whileFTI treatment almost totally abolishes it, and leads to relocalizationof progerin into nucleoplasmic aggregates (d); (b) unprocessedprogerin-SSIM mimics the phenotype of FTI-treated cells expressingauthentic progerin-CSIM, with all progerin relocalized intonucleoplasmic aggregates, and this does not change significantly withtreatment (e); (c) progerin-CSIL, predicted to be geranylgeranylated,recreates nuclear blebbing but is resistant to FTI treatment (f). Cellswere visualized 48 hours after transfection and photomicrographs weretaken at 60× magnification.

FIG. 24: The percentage of blebbed HeLa nuclei when transientlytransfected with the above four constructs and treated with a singledose of lonafarnib/SCH66336 at the time of transfection. Percentageswere read by three independent observers, who scored 200 cells for eachexperiment and were blind to the construct being used. Error bars showthe standard error of the mean, and the p-values are for the comparisonof the treated cells to the untreated control. Wild-type lamin A-CSIMdoes not change significantly from the typical 5% nuclear blebbingpercentage of normal cells. The progerin-CSIM shows a dramaticdose-response. The progerin-CSIL, expected to be geranylgeranylated, isresistant to inhibition of farnesylation. The progerin-SSIM, whichcannot be farnesylated or geranylgeranylated, demonstrates virtually noblebbing.

FIG. 25: FTI treatment causes reversion of the nuclear blebbing in twodifferent progerin-expressing HGPS human fibroblasts. Cells were stainedwith anti-lamin A antibody after being treated for 3 days with either 0,1, or 2 μM of the FTI lonafarnib/SCH66336 (60×).

FIG. 26: The percentage of blebbed nuclei when HGPS skin fibroblastswere treated once daily for three days with the above doses oflonafarnib. AG06299 is from the 34 year-old unaffected mother (passage31) of AG06917, an affected 6 year-old (passage 14). AG11513 is from anaffected 8 year-old (passage 7), and AG11498 is from an affected 14year-old (passage 11). Percentages were read by three independentobservers, who scored 200 cells for each experiment and were blind tothe genotype of the fibroblasts and the dose of the FTI being used.Error bars show the standard error of the mean, and the p-values are forthe comparison of the treated cells to the untreated control.

FIG. 27: FTI treatment alone is sufficient to impair progerinprocessing. NIH-3T3 cells transiently expressing empty vector (“v.o.”),or GFP-tagged progerin-CSIM (“Progerin”) or GFP-tagged progerin-SSIM(“SSIM”) were treated for 48 hours with vehicle (DMSO) or inhibitors ofFTase or GGTase I (5 μM FTI-2153 or GGTI-2166, respectively). Total celllysates were resolved on SDS-PAGE and immunoblotted with anti-GFP todetect GFP-progerin-CSIM or the fully unprocessed mutantGFP-progerin-SSIM. Treatment with FTI, but not GGTI, induces a mobilityshift of progerin-CSIM to that of the unprocessed progerin-SSIM,demonstrating that progerin is a FTI target that does not undergoalternative prenylation and modification by GGTase I. Schematic diagramsat the top of each lane indicate the molecular entity that constitutesthe major species in each experiment. P=progerin sequence up to CAAXmotif (SEQ ID NO: 31); C=cysteine; F=farnesyl group.

SEQUENCE LISTING

The nucleic and amino acid sequences listed in the accompanying sequencelisting are shown using standard letter abbreviations for nucleotidebases, and three letter code for amino acids, as defined in 37 C.F.R.1.822. Only one strand of each nucleic acid sequence is shown, but thecomplementary strand is understood as included by any reference to thedisplayed strand. In the accompanying sequence listing:

SEQ ID NOs: 1 and 2 are primers (laminAF1 and laminAR1, respectively)used to amplify normal and mutant lamin A/C.

SEQ ID NOs: 3-8 are primers used to introduce the L647R mutant into thepRevTRE-GFP-lamin A construct.

SEQ ID NOs: 9 and 10 are a pair of forward and reverse primers.

SEQ ID NOs: 11-18 are primers used to generate lamin A CAAX mutants. SEQID NO: 19 is the CSIM Human lamin A (LMNA) wt mRNA coding sequence(nucleotide residues 213-2207 of GenBank Accession: NM_(—)170707).

SEQ ID NO: 20 is the CSIM LMNA protein translation.

SEQ ID NO: 21 is the CSIL Human lamin A (LMNA) wt mRNA coding sequence(nucleotide residues 213-2207 of GenBank Accession: NM_(—)170707).

SEQ ID NO: 22 is the CSIL LMNA protein translation.

SEQ ID NO: 23 is the SSIM Human lamin A (LMNA) wt mRNA coding sequence(nucleotide residues 213-2207 of GenBank Accession: NM_(—)170707).

SEQ ID NO: 24 is the SSIM LMNA protein translation.

SEQ ID NO: 25 is the CSIM Human lamin A (LMNA) del 150 mRNA codingsequence.

SEQ ID NO: 26 is the CSIM LMNA_del50 protein translation.

SEQ ID NO: 27 is the Human lamin A (LMNA) CSIL del 150 mRNA codingsequence.

SEQ ID NO: 28 is the CSIL LMNA_del50 protein translation.

SEQ ID NO: 29 is the SSIM Human lamin A (LMNA) del 150 mRNA codingsequence.

SEQ ID NO: 30 is the SSIM LMNA_del50 protein translation.

SEQ ID NOs: 31-34 are the CAAX box motif of the prelamin protein andvariants thereof.

DETAILED DESCRIPTION I. Abbreviations

EC: endothelial cells

EGFP: enhanced green fluorescent protein

FPP: farnesyl pyrophosphate

FTase: farnesyltransferase

FTI: farnesyltransferase inhibitor

GFP: green fluorescent protein

GGTase I: geranylgeranyltransferase

GGTI: geranylgeranyltransferase inhibitor

HGPS: Hutchinson-Gilford Progeria Syndrome

ICC: immunocytochemistry

LA: lamin A

LMNA: gene encoding lamin A and lamin C

MADB: mandibuloacral dysplasia

MG: minigene

ORF: open reading frame

PD: Passage doubling

RT-PCR: reverse-transcription polymerase chain reaction

UTRs: untranslated regions

VSMC: vascular smooth muscle cells

II. Terms

Unless otherwise noted, technical terms are used according toconventional usage. Definitions of common terms in molecular biology maybe found in Benjamin Lewin, Genes V, published by Oxford UniversityPress, 1994 (ISBN 0-19-854287-9); Kendrew et al. (eds.), TheEncyclopedia of Molecular Biology, published by Blackwell Science Ltd.,1994 (ISBN 0-632-02182-9); and Robert A. Meyers (ed.), Molecular Biologyand Biotechnology: a Comprehensive Desk Reference, published by VCHPublishers, Inc., 1995 (ISBN 1-56081-569-8).

In order to facilitate review of the various embodiments of theinvention, we provide the following information concerning some of theterms used herein. Additional information may be found in theliterature, including the references cited and incorporated by referenceherein.

Abnormal: Deviation from normal characteristics. Normal characteristicscan be found in a control, a standard for a population, etc. Forinstance, where the abnormal condition is a disease condition, such asprogeria, a few appropriate sources of normal characteristics mightinclude an individual who is not suffering from the disease (e.g.,progeria), a population standard of individuals believed not to besuffering from the disease, etc.

Likewise, abnormal may refer to a condition that is associated with adisease. The term “associated with” includes an increased risk ofdeveloping the disease as well as the disease itself. For instance, acertain abnormality (such as an abnormality in an LMNA nucleic acid orLamin protein expression) can be described as being associated with thebiological conditions of progeria and tendency to develop prematureaging disease or condition.

An abnormal nucleic acid, such as an abnormal LMNA nucleic acid, is onethat is different in some manner to a normal (wildtype) nucleic acid.Such abnormality includes but is not necessarily limited to: (1) amutation in the nucleic acid (such as a point mutation (e.g., a singlenucleotide polymorphism) or short deletion or duplication of a few toseveral nucleotides); (2) a mutation in the control sequence(s)associated with that nucleic acid such that replication or expression ofthe nucleic acid is altered (such as the functional inactivation of apromoter); (3) a decrease in the amount or copy number of the nucleicacid in a cell or other biological sample (such as a deletion of thenucleic acid, either through selective gene loss or by the loss of alarger section of a chromosome or under expression of the mRNA); (4) anincrease in the amount or copy number of the nucleic acid in a cell orsample (such as a genomic amplification of part or all of the nucleicacid or the overexpression of an mRNA), each compared to a control orstandard; and (5) an alteration in a sequence that controls the splicingmechanism, in such a way that either a normal splice signal isinactivated or an abnormal splice signal is created. It will beunderstood that these types of abnormalities can co-exist in the samenucleic acid or in the same cell or sample; for instance, agenomic-amplified nucleic acid sequence may also contain one or morepoint mutations. In addition, it is understood that an abnormality in anucleic acid may be associated with, and in fact may cause, anabnormality in expression of the corresponding protein.

Abnormal protein expression, such as abnormal lamin A proteinexpression, refers to expression of a protein that is in some mannerdifferent to expression of the protein in a normal (wildtype) situation.This includes but is not necessarily limited to: (1) a mutation in theprotein such that one or more of the amino acid residues is different;(2) a short deletion or addition of one or a few amino acid residues tothe sequence of the protein; (3) a longer deletion or addition of aminoacid residues, such that an entire protein domain or sub-domain isremoved or added; (4) expression of an increased amount of the protein,compared to a control or standard amount; (5) expression of an decreasedamount of the protein, compared to a control or standard amount; (6)alteration of the subcellular localization or targeting of the protein;(7) alteration of the temporally regulated expression of the protein(such that the protein is expressed when it normally would not be, oralternatively is not expressed when it normally would be); and (8)alteration of the localized (e.g., organ or tissue specific) expressionof the protein (such that the protein is not expressed where it wouldnormally be expressed or is expressed where it normally would not beexpressed), each compared to a control or standard.

One particular type of protein abnormality that is contemplated hereininvolves changes in the post-translational processing of a protein. Aprotein that is abnormally post-translationally processed includes, forinstance, proteins that processed in a different way, or to a differentextent, than the wild-type (normal) version of the protein. Progerin isan example of an abnormally processed protein; it is not proteolyticallyprocessed in the same way as the corresponding wild-type protein, laminA. In addition, progerin is abnormal in that it remains farnesylatedafter its processing is completed (to the extent that a cell expressinga nucleic acid encoding progerin processes the protein). This can bereferred to as being “constitutively” farnesylated, in that anindividual progerin protein remains farnesylated in the context of anotherwise unaltered cell. In contrast, a “normal” lamin A protein isonly transiently farnesylated, because full post-translationalprocessing of lamin A eventually results in loss of the farnesyl groupfrom the mature protein (by way of a required proteolytic cleavagestep). Progerin can be considered an abnormally farnesylated form oflamin A; other abnormally farnesylated forms of lamins are discussedherein, including forms engineered to lack a proteolytic cleavage site,to lack the farnesylation site, and so forth. Other potential “abnormal”post-translational processing includes changes in other added groups(e.g., methylations, phosphorylations, glycosylations, and so forth),alterations in proteolytic processing, and so forth.

Controls or standards appropriate for comparison to a sample, for thedetermination of abnormality, include samples believed to be normal aswell as laboratory determined values, even though such values arepossibly arbitrarily set, and keeping in mind that such values may varyfrom laboratory to laboratory. Laboratory standards and values may beset based on a known or determined population value and may be suppliedin the format of a graph or table that permits easy comparison ofmeasured, experimentally determined values.

Analog, derivative or mimetic: An analog is a molecule that differs inchemical structure from a parent or reference compound, for example ahomolog (differing by an incremental change in the chemical structure,such as a difference in the length of an alkyl chain), a molecularfragment, a structure that differs by one or more functional groups, achange in ionization. Structural analogs are often found usingquantitative structure activity relationships (QSAR), with techniquessuch as those disclosed in Remington (The Science and Practice ofPharmacology, 19th Edition (1995), chapter 28). A derivative is asubstance related to a base structure, and theoretically derivable fromthe base structure. A mimetic is a biomolecule that mimics the activityof another biologically active molecule. Biologically active moleculescan include chemical structures that mimic the biological activities ofa compound, for instance a native siRNA.

Array: An arrangement of molecules, particularly biologicalmacromolecules (such as polypeptides or nucleic acids) in addressablelocations on a substrate. The array may be regular (arranged in uniformrows and columns, for instance) or irregular. The number of addressablelocations on the array can vary, for example from a few (such as three)to more than 50, 100, 200, 500, 1000, 10,000, or more. A “microarray” isan array that is miniaturized so as to require or benefit frommicroscopic examination, or other magnification, for its evaluation.Further miniaturization can be used to produce “nanoarrays.”

Within an array, each arrayed molecule sample is addressable, in thatits location can be reliably and consistently determined within at leasttwo dimensions of the array surface. In ordered arrays, the location ofeach molecule sample can be assigned to the sample at the time when itis spotted or otherwise applied onto the array surface, and a key may beprovided in order to correlate each location with the appropriatetarget. Often, ordered arrays are arranged in a symmetrical gridpattern, but samples could be arranged in other patterns (e.g., inradially distributed lines, spiral lines, or ordered clusters).Addressable arrays are computer readable, in that a computer can beprogrammed to correlate a particular address on the array withinformation (such as hybridization or binding data, including forinstance signal intensity). In some examples of computer readableformats, the individual “spots” on the array surface will be arrangedregularly in a pattern (e.g., a Cartesian grid pattern) that can becorrelated to address information by a computer.

The sample application “spot” on an array may assume many differentshapes. Thus, though the term “spot” is used, it refers generally to alocalized deposit of nucleic acid, and is not limited to a round orsubstantially round region. For instance, substantially square regionsof mixture application can be used with arrays encompassed herein, ascan be regions that are substantially rectangular (such as a slotblot-type application), or triangular, oval, or irregular. The shape ofthe array substrate itself is also immaterial, though it is usuallysubstantially flat and may be rectangular or square in general shape.

DNA (deoxyribonucleic acid): DNA is a long chain polymer which comprisesthe genetic material of most living organisms (some viruses have genescomprising ribonucleic acid (RNA)). The repeating units in DNA polymersare four different nucleotides, each of which comprises one of the fourbases, adenine, guanine, cytosine and thymine bound to a deoxyribosesugar to which a phosphate group is attached. Triplets of nucleotides(referred to as codons) code for each amino acid in a polypeptide, orfor a stop signal. The term codon is also used for the corresponding(and complementary) sequences of three nucleotides in the mRNA intowhich the DNA sequence is transcribed.

Unless otherwise specified, any reference to a DNA molecule is intendedto include the reverse complement of that DNA molecule. Except wheresingle-strandedness is required by the text herein, DNA molecules,though written to depict only a single strand, encompass both strands ofa double-stranded DNA molecule. Thus, a reference to the nucleic acidmolecule that encodes lamin A, or a fragment thereof, encompasses boththe sense strand and its reverse complement. Thus, for instance, it isappropriate to generate probes or primers from the reverse complementsequence of the disclosed nucleic acid molecules.

Deletion: The removal of a sequence of DNA, the regions on either sideof the removed sequence being joined together.

Epitope tags are short stretches of amino acids to which a specificantibody can be raised, which in some embodiments allows one tospecifically identify and track the tagged protein that has been addedto a living organism or to cultured cells. Detection of the taggedmolecule can be achieved using a number of different techniques.Examples of such techniques include: immunohistochemistry,immunoprecipitation, flow cytometry, immunofluorescence microscopy,ELISA, immunoblotting (“western”), and affinity chromatography. Examplesof useful epitope tags include but are not limited to FLAG, T7, HA(hemagglutinin) and myc.

Feature: An addressable spot/element containing a molecule or mixture ofmolecules on an array. Features may be created by printing themolecule(s), usually within some type of matrix, onto the array platformby a printing device, such as a quill-like pen, or by a touch-lessdeposition system (see, e.g., Harris et al., Nature Biotech. 18:384-385,2000). Alternatively, in some embodiments the feature can be made by insitu synthesis of the molecule on the array substrate.

Fluorescent protein: A protein that either directly (through itsprimary, secondary, or tertiary structure) or indirectly (through aco-factor, non-protein chromophore, or a substrate, or due to theaddition of a fluor) produces or emits fluorescent light. Non-limitingexamples of fluorescent proteins are the green fluorescent protein (GFP;see, for instance, GenBank Accession Number M62654) from the PacificNorthwest jellyfish, Aequorea victoria and natural and engineeredvariants thereof (see, for instance, U.S. Pat. Nos. 5,804,387;6,090,919; 6,096,865; 6,054,321; 5,625,048; 5,874,304; 5,777,079;5,968,750; 6,020,192; and 6,146,826; and published international patentapplication WO 99/64592).

Fluorophore: A chemical compound, which when excited by exposure to aparticular wavelength of light, emits light (i.e., fluoresces), forexample at a different wavelength. Fluorophores can be described interms of their emission profile, or “color.” Green fluorophores, forExampleCY3® green fluorophore, FITC, and OREGON GREEN® greenfluorophore, are characterized by their emission at wavelengthsgenerally in the range of 515-540λ. Red fluorophores, for example TEXASRED® red fluorophore, CY5® red fluorophore and tetramethylrhodamine, arecharacterized by their emission at wavelengths generally in the range of590-690λ.

Examples of fluorophores that may be used are provided in U.S. Pat. No.5,866,366 to Nazarenko et al., and include for instance:4-acetamido-4′-isothiocyanatostilbene-2,2′disulfonic acid, acridine andderivatives such as acridine and acridine isothiocyanate,5-(2′-aminoethyl)aminonaphthalene-1-sulfonic acid (EDANS),4-amino-N-[3-vinylsulfonyl)phenyl]naphthalimide-3,5 disulfonate (LuciferYellow VS), N-(4-anilino-1-naphthyl)maleimide, anthranilamide, BrilliantYellow, coumarin and derivatives such as coumarin,7-amino-4-methylcoumarin (AMC, Coumarin 120),7-amino-4-trifluoromethylcouluarin (Coumaran 151); cyanosine;4′,6-diaminidino-2-phenylindole (DAPI);5′,5″-dibromopyrogallol-sulfonephthalein (Bromopyrogallol Red);7-diethylamino-3-(4′-isothiocyanatophenyl)-4-methylcoumarin;diethylenetriamine pentaacetate;4,4′-diisothiocyanatodihydro-stilbene-2,2′-disulfonic acid;4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid;5-[dimethylamino]naphthalene-1-sulfonyl chloride (DNS, dansyl chloride);4-(4′-dimethylaminophenylazo)benzoic acid (DABCYL);4-dimethylaminophenylazophenyl-4′-isothiocyanate (DABITC); eosin andderivatives such as eosin and eosin isothiocyanate; erythrosin andderivatives such as erythrosin B and erythrosin isothiocyanate;ethidium; fluorescein and derivatives such as 5-carboxyfluorescein(FAM), 5-(4,6-dichlorotriazin-2-yl)aminofluorescein (DTAF),2′7′-dimethoxy-4′5′-dichloro-6-carboxyfluorescein (JOE), fluorescein,fluorescein isothiocyanate (FITC), and QFITC (XRITC); fluorescamine;IR144; IR1446; Malachite Green isothiocyanate; 4-methylumbelliferone;ortho cresolphthalein; nitrotyrosine; pararosaniline; Phenol Red;B-phycoerythrin; o-phthaldialdehyde; pyrene and derivatives such aspyrene, pyrene butyrate and succinimidyl 1-pyrene butyrate; Reactive Red4 (Cibacron® Brilliant Red 3B-A); rhodamine and derivatives such as6-carboxy-X-rhodamine (ROX), 6-carboxyrhodamine (R6G), lissaminerhodamine B sulfonyl chloride, rhodamine (Rhod), rhodamine B, rhodamine123, rhodamine X isothiocyanate, sulforhodamine B, sulforhodamine 101and sulfonyl chloride derivative of sulforhodamine 101 (TEXAS RED® redfluorophore); N,N,N′,N′-tetramethyl-6-carboxyrhodamine (TAMRA);tetramethyl rhodamine; tetramethyl rhodamine isothiocyanate (TRITC);riboflavin; rosolic acid and terbium chelate derivatives.

Other contemplated fluorophores include GFP (green fluorescent protein),Lissamine™, diethylaminocoumarin, fluorescein chlorotriazinyl,naphthofluorescein, 4,7-dichlororhodamine and xanthene and derivativesthereof. Other fluorophores known to those skilled in the art may alsobe used.

Inhibiting or treating a disease: Inhibiting the full development of adisease, disorder or condition, for example, in a subject who is at riskfor a disease such as a laminopathy, an aging-associated disease orcondition, atherosclerosis or cardiovascular disease. “Treatment” refersto a therapeutic intervention that ameliorates a sign or symptom of adisease or pathological condition after it has begun to develop. As usedherein, the term “ameliorating,” with reference to a disease,pathological condition or symptom, refers to any observable beneficialeffect of the treatment. The beneficial effect can be evidenced, forexample, by a delayed onset of clinical symptoms of the disease in asusceptible subject, a reduction in severity of some or all clinicalsymptoms of the disease, a slower progression of the disease, areduction in the number of relapses of the disease, an improvement inthe overall health or well-being of the subject, or by other parameterswell known in the art that are specific to the particular disease orcondition.

Injectable composition: A pharmaceutically acceptable fluid compositionincluding at least one active ingredient (for instance, a dsRNA). Theactive ingredient is usually dissolved or suspended in a physiologicallyacceptable carrier, and the composition can additionally include minoramounts of one or more non-toxic auxiliary substances, such asemulsifying agents, preservatives, and pH buffering agents and the like.Such injectable compositions that are useful for use with the providednucleotides and proteins are conventional; appropriate formulations arewell known in the art.

Isolated: An “isolated” biological component (such as a nucleic acidmolecule, protein or organelle) has been substantially separated orpurified away from other biological components in the cell of theorganism in which the component naturally occurs, e.g., otherchromosomal and extra-chromosomal DNA and RNA, proteins and organelles.Nucleic acids and proteins that have been “isolated” include nucleicacids and proteins purified by standard purification methods. The termalso embraces nucleic acids and proteins prepared by recombinantexpression in a host cell as well as chemically synthesized nucleicacids.

Label: A composition detectable by (for instance) spectroscopic,photochemical, biochemical, immunochemical, or chemical means. Typicallabels include fluorescent proteins or protein tags, fluorophores,radioactive isotopes (including for instance ³²P), ligands, biotin,digoxigenin, chemiluminescent agents, electron-dense reagents (such asmetal sols and colloids), and enzymes (e.g., for use in an ELISA),haptens, and proteins or peptides (such as epitope tags) for whichantisera or monoclonal antibodies are available. Methods for labelingand guidance in the choice of labels useful for various purposes arediscussed, e.g., in Sambrook et al., in Molecular Cloning: A LaboratoryManual, Cold Spring Harbor Laboratory Press (1989) and Ausubel et al.,in Current Protocols in Molecular Biology, John Wiley & Sons, New York(1998). A label often generates a measurable signal, such asradioactivity, fluorescent light or enzyme activity, which can be usedto detect and/or quantitate the amount of labeled molecule.

Nucleotide: “Nucleotide” includes, but is not limited to, a monomer thatincludes a base linked to a sugar, such as a pyrimidine, purine orsynthetic analogs thereof, or a base linked to an amino acid, as in apeptide nucleic acid (PNA). A nucleotide is one monomer in apolynucleotide. A nucleotide sequence refers to the sequence of bases ina polynucleotide.

Oligonucleotide: An oligonucleotide is a plurality of joined nucleotidesjoined by native phosphodiester bonds, between about 6 and about 300nucleotides in length. An oligonucleotide analog refers to moieties thatfunction similarly to oligonucleotides but have non-naturally occurringportions. For example, oligonucleotide analogs can contain non-naturallyoccurring portions, such as altered sugar moieties or inter-sugarlinkages, such as a phosphorothioate oligodeoxynucleotide. Functionalanalogs of naturally occurring polynucleotides can bind to RNA or DNA,and include peptide nucleic acid (PNA) molecules.

Particular oligonucleotides and oligonucleotide analogs can includelinear sequences up to about 200 nucleotides in length, for example asequence (such as DNA or RNA) that is at least 6 bases, for example atleast 8, 10, 15, 20, 25, 30, 35, 40, 45, 50, 100 or even 200 bases long,or from about 6 to about 50 bases, for example about 10-25 bases, suchas 12, 15 or 20 bases.

Operably linked: A first nucleic acid sequence is operably linked with asecond nucleic acid sequence when the first nucleic acid sequence isplaced in a functional relationship with the second nucleic acidsequence. For instance, a promoter is operably linked to a codingsequence if the promoter affects the transcription or expression of thecoding sequence. Generally, operably linked DNA sequences are contiguousand, where necessary to join two protein-coding regions, in the samereading frame.

Open reading frame: A series of nucleotide triplets (codons) coding foramino acids without any internal termination codons. These sequences areusually translatable into a peptide.

Parenteral: Administered outside of the intestine, e.g., not via thealimentary tract. Generally, parenteral formulations are those that willbe administered through any possible mode except ingestion. This termespecially refers to injections, whether administered intravenously,intrathecally, intramuscularly, intraperitoneally, or subcutaneously,and various surface applications including intranasal, intradermal, andtopical application, for instance.

Peptide Nucleic Acid (PNA): An oligonucleotide analog with a backbonecomprised of monomers coupled by amide (peptide) bonds, such as aminoacid monomers joined by peptide bonds.

Pharmaceutically acceptable carriers: The pharmaceutically acceptablecarriers useful with this disclosure are conventional. Martin,Remington's Pharmaceutical Sciences, published by Mack Publishing Co.,Easton, Pa., 19th Edition, 1995, describes compositions and formulationssuitable for pharmaceutical delivery of the nucleotides and proteinsherein disclosed.

In general, the nature of the carrier will depend on the particular modeof administration being employed. For instance, parenteral formulationsusually comprise injectable fluids that include pharmaceutically andphysiologically acceptable fluids such as water, physiological saline,balanced salt solutions, aqueous dextrose, glycerol or the like as avehicle. For solid compositions (e.g., powder, pill, tablet, or capsuleforms), conventional non-toxic solid carriers can include, for example,pharmaceutical grades of mannitol, lactose, starch, or magnesiumstearate. In addition to biologically-neutral carriers, pharmaceuticalcompositions to be administered can contain minor amounts of non-toxicauxiliary substances, such as wetting or emulsifying agents,preservatives, and pH buffering agents and the like, for example sodiumacetate or sorbitan monolaurate.

Polymorphism: Variant in a sequence of a gene. Polymorphisms can bethose variations (nucleotide sequence differences) that, while having adifferent nucleotide sequence, produce functionally equivalent geneproducts, such as those variations generally found between individuals,different ethnic groups, geographic locations. The term polymorphismalso encompasses variations that produce gene products with alteredfunction, e.g., variants in the gene sequence that lead to gene productsthat are not functionally equivalent. This term also encompassesvariations that produce no gene product, an inactive gene product, orincreased gene product. The term polymorphism may be usedinterchangeably with allele or mutation, unless context clearly dictatesotherwise.

Polymorphisms can be referred to, for instance, by the nucleotideposition at which the variation exists, by the change in amino acidsequence caused by the nucleotide variation, or by a change in someother characteristic of the nucleic acid molecule that is linked to thevariation (e.g., an alteration of a secondary structure such as astem-loop, or an alteration of the binding affinity of the nucleic acidfor associated molecules, such as polymerases, RNases, and so forth). Inthe current instance, Mutation 1 is also referred to as G608G(GGC>GGT),indicating that the mutation is in codon 608, that it is silent (in thatit causes no change in the encoded amino acid), and that the exactnucleotide sequence change is C to T in the third position of the codon.Similarly, Mutation 2 is also referred to as G608S(GGC>AGC), indicatingthat the mutation is in codon 608, that it causes an amino acidsubstitution (glycine to serine), and that the exact nucleotide sequencechange is G to A in the first position of the codon.

Probes and primers: A probe comprises an isolated nucleic acid attachedto a detectable label or other reporter molecule. Typical labels includeradioactive isotopes, enzyme substrates, co-factors, ligands,chemiluminescent or fluorescent agents, haptens, and enzymes. Methodsfor labeling and guidance in the choice of labels appropriate forvarious purposes are discussed, e.g., in Sambrook et al. (In MolecularCloning: A Laboratory Manual, CSHL, New York, 1989) and Ausubel et al.(In Current Protocols in Molecular Biology, John Wiley & Sons, New York,1998).

Primers are short nucleic acid molecules, for instance DNAoligonucleotides 10 nucleotides or more in length, for example thathybridize to contiguous complementary nucleotides or a sequence to beamplified. Longer DNA oligonucleotides may be about 15, 20, 25, 30 or 50nucleotides or more in length. Primers can be annealed to acomplementary target DNA strand by nucleic acid hybridization to form ahybrid between the primer and the target DNA strand, and then the primerextended along the target DNA strand by a DNA polymerase enzyme. Primerpairs can be used for amplification of a nucleic acid sequence, e.g., bythe polymerase chain reaction (PCR) or other nucleic-acid amplificationmethods known in the art. Other examples of amplification include stranddisplacement amplification, as disclosed in U.S. Pat. No. 5,744,311;transcription-free isothermal amplification, as disclosed in U.S. Pat.No. 6,033,881; repair chain reaction amplification, as disclosed in WO90/01069; ligase chain reaction amplification, as disclosed in EP-A-320308; gap filling ligase chain reaction amplification, as disclosed inU.S. Pat. No. 5,427,930; and NASBA™ RNA transcription-freeamplification, as disclosed in U.S. Pat. No. 6,025,134.

Nucleic acid probes and primers can be readily prepared based on thenucleic acid molecules provided in this disclosure. It is alsoappropriate to generate probes and primers based on fragments orportions of these disclosed nucleic acid molecules, for instance regionsthat encompass the identified polymorphisms at nucleotide 1822 andnucleotide 1824 within the LMNA coding sequence.

Methods for preparing and using nucleic acid probes and primers aredescribed, for example, in Sambrook et al. (In Molecular Cloning: ALaboratory Manual, CSHL, New York, 1989), Ausubel et al. (ed.) (InCurrent Protocols in Molecular Biology, John Wiley & Sons, New York,1998), and Innis et al. (PCR Protocols, A Guide to Methods andApplications, Academic Press, Inc., San Diego, Calif., 1990).Amplification primer pairs can be derived from a known sequence, forexample, by using computer programs intended for that purpose such asPrimer (Version 0.5, © 1991, Whitehead Institute for BiomedicalResearch, Cambridge, Mass.). One of ordinary skill in the art willappreciate that the specificity of a particular probe or primerincreases with its length. Thus, for example, a primer comprising 30consecutive nucleotides of a lamin A-encoding nucleotide or flankingregion thereof (a “lamin A primer” or “lamin A probe”) will anneal to atarget sequence with a higher specificity than a corresponding primer ofonly 15 nucleotides. Thus, in order to obtain greater specificity,probes and primers can be selected that comprise at least 20, 25, 30,35, 40, 45, 50 or more consecutive nucleotides of a lamin A nucleotidesequences.

The disclosure thus includes isolated nucleic acid molecules thatcomprise specified lengths of the lamin A encoding sequence and/orflanking regions. Such molecules may comprise at least about 10, 15, 20,23, 25, 30, 35, 40, 45 or 50 consecutive nucleotides of these sequencesor more, and may be obtained from any region of the disclosed sequences.By way of example, the human LMNA locus, cDNA, ORF, coding sequence andgene sequences (including sequences both upstream and downstream of theLMNA coding sequence) may be apportioned into about halves or quartersbased on sequence length, and the isolated nucleic acid molecules (e.g.,oligonucleotides) may be derived from the first or second halves of themolecules, or any of the four quarters. The cDNA also could be dividedinto smaller regions, e.g. about eighths, sixteenths, twentieths,fiftieths and so forth, with similar effect.

In particular embodiments, isolated nucleic acid molecules comprise oroverlap at least one residue position designated as being associatedwith a polymorphism that is predictive of progeria and/or a prematureaging disease or condition. Such polymorphism sites include position1822 (corresponding to the Mutation 2 polymorphism) and position 1824(corresponding to the Mutation 1 polymorphism).

Protein: A biological molecule, particularly a polypeptide, expressed bya gene and comprised of amino acids.

Purified: The term “purified” does not require absolute purity; rather,it is intended as a relative term. Thus, for example, a purified proteinpreparation is one in which the protein referred to is more pure thanthe protein in its natural environment within a cell or within aproduction reaction chamber (as appropriate).

Recombinant: A recombinant nucleic acid is one that has a sequence thatis not naturally occurring or has a sequence that is made by anartificial combination of two otherwise separated segments of sequence.This artificial combination can be accomplished by chemical synthesisor, more commonly, by the artificial manipulation of isolated segmentsof nucleic acids, e.g., by genetic engineering techniques.

Specific binding agent: An agent that binds substantially only to adefined target. Thus a lamin A protein-specific binding agent bindssubstantially only the lamin A protein. As used herein, the term “Laminprotein specific binding agent” includes anti-Lamin protein antibodies(and functional fragments thereof) and other agents (such as solublereceptors) that bind substantially only to a Lamin protein. It isparticularly contemplated in specific embodiments that certainLamin-specific binding agents are specific for one form of Lamin, suchas lamin A or lamin C.

Anti-Lamin protein antibodies may be produced using standard proceduresdescribed in a number of texts, including Harlow and Lane (Antibodies, ALaboratory Manual, CSHL, New York, 1988). The determination that aparticular agent binds substantially only to the target protein mayreadily be made by using or adapting routine procedures. One suitable invitro assay makes use of the Western blotting procedure (described inmany standard texts, including Harlow and Lane (Antibodies, A LaboratoryManual, CSHL, New York, 1988)). Western blotting may be used todetermine that a given target protein binding agent, such as ananti-lamin A protein monoclonal antibody, binds substantially only tothe specified target protein.

Shorter fragments of antibodies can also serve as specific bindingagents. For instance, FAbs, Fvs, and single-chain Fvs (SCFvs) that bindto lamin A would be lamin A-specific binding agents. These antibodyfragments are defined as follows: (1) FAb, the fragment which contains amonovalent antigen-binding fragment of an antibody molecule produced bydigestion of whole antibody with the enzyme papain to yield an intactlight chain and a portion of one heavy chain; (2) FAb′, the fragment ofan antibody molecule obtained by treating whole antibody with pepsin,followed by reduction, to yield an intact light chain and a portion ofthe heavy chain; two FAb′ fragments are obtained per antibody molecule;(3) (FAb′)₂, the fragment of the antibody obtained by treating wholeantibody with the enzyme pepsin without subsequent reduction; (4)F(Ab′)₂, a dimer of two FAb′ fragments held together by two disulfidebonds; (5) Fv, a genetically engineered fragment containing the variableregion of the light chain and the variable region of the heavy chainexpressed as two chains; and (6) single chain antibody (“SCA”), agenetically engineered molecule containing the variable region of thelight chain, the variable region of the heavy chain, linked by asuitable polypeptide linker as a genetically fused single chainmolecule. Methods of making these fragments are routine.

Subject: Living multi-cellular vertebrate organisms, a category thatincludes both human and non-human mammals. This term encompasses bothknown and unknown individuals, such that there is no requirement that aperson working with a sample from a subject know who the subject is, oreven from where the sample was acquired.

Subject susceptible to a disease or condition: A subject capable of,prone to, or predisposed to developing a disease or condition. It isunderstood that a subject already having or showing symptoms of adisease or condition is considered “susceptible” since they have alreadydeveloped it.

Therapeutically effective dose: A dose sufficient to preventadvancement, or to cause regression of the disease, or which is capableof relieving symptoms caused by the disease, such as a laminopathy orage related disease or condition (for instance, atherosclerosis). Forexample, a therapeutically effective amount of an inhibitor can varyfrom about 0.1 nM per kilogram (kg) body weight to about 1 μM per kgbody weight, such as about 1 nM to about 500 nM per kg body weight, orabout 5 nM to about 50 nM per kg body weight. The exact dose is readilydetermined by one of skill in the art based on the potency of thespecific compound, the age, weight, sex and physiological condition ofthe subject.

Transfected or Transformed: A process by which a nucleic acid moleculeis introduced into live cells, for instance by molecular biologytechniques, resulting in a transfected (or transformed) cell. As usedherein, the term transfection encompasses all techniques by which anucleic acid molecule might be introduced into such a cell, includingtransduction with viral vectors, transfection with plasmid vectors, andintroduction of DNA by electroporation, lipofection, and particle gunacceleration.

Vector: A nucleic acid molecule as introduced into a host cell, therebyproducing a transfected host cell. Recombinant DNA vectors are vectorshaving recombinant DNA. A vector can include nucleic acid sequences thatpermit it to replicate in a host cell, such as an origin of replication.A vector can also include one or more selectable marker genes and othergenetic elements known in the art. Viral vectors are recombinant DNAvectors having at least some nucleic acid sequences derived from one ormore viruses.

Unless otherwise explained, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which this invention belongs. The singular terms“a,” “an,” and “the” include plural referents unless context clearlyindicates otherwise. Similarly, the word “or” is intended to include“and” unless the context clearly indicates otherwise. Hence “comprisingA or B” means including A, or B, or A and B. It is further to beunderstood that all base sizes or amino acid sizes, and all molecularweight or molecular mass values, given for nucleic acids or polypeptidesare approximate, and are provided for description. Although methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, suitable methods andmaterials are described below. All publications, patent applications,patents, and other references mentioned herein are incorporated byreference in their entirety. In case of conflict, the presentspecification, including explanations of terms, will control. Inaddition, the materials, methods, and examples are illustrative only andnot intended to be limiting.

III. Overview of Several Embodiments

A first embodiment is a method of reducing at least one cellular defectin a cell from a subject susceptible to a disease or conditioncharacterized by farnesylation of an abnormally farnesylated form of alamin, comprising administering to the cell a therapeutically effectivedose of a farnesyltransferase inhibitor (FTI). In examples of suchmethods, the cellular defect involves mis-localization of a farnesylatedlamin, mis-localization of a non-farnesylated lamin, nuclear membranedisruption, aggregation of lamin, nuclear lobulation, nuclear blebbing,cytoskeleton disruption, early senescence, apoptosis, reduced secretionof MMP-3, or two or more thereof.

Examples of diseases or conditions characterized by farnesylation of anabnormally farnesylated form of a lamin include laminopathies, whichinclude but are not limited to Hutchinson-Gilford Progeria Syndrome(HGPS), progeria, muscular dystrophy, Charcot-Marie-Tooth disorder, andWerner syndrome.

There is also provided a method of treating a subject having orpredisposed to a laminopathy, which method involves administering to thesubject an effective amount of at least one FTI.

Yet another provided method is a method of preventing progression of alaminopathy in a subject having or predisposed to the laminopathy, whichmethod involves administering at least one FTI to the subject.

In examples of the various embodiments, the normally non-farnesylatedlamin is a lamin other than Lamin B. For instance, in specificinstances, the normally non-farnesylated lamin is lamin A.

It is specifically contemplated herein that the subject of any of theprovided methods may be a subject having, or suspected of having, orsusceptible to, atherosclerosis.

By way of specific, non-limiting examples, the FTI used in any methodprovided herein can be PD169541, R115777, SCH66336, or afarnesyltransferase inhibitory derivative thereof. Optionally, in any ofthe methods, a second therapeutic compound can be administered to thecell or subject. Examples of such second therapeutic compounds includeanother FTI, or a non-FTI compound.

A further embodiment is a method of screening for a compound useful intreating a laminopathy in a mammal. In an example of this embodiment,the method involves determining if a test compound alters a cellulardefect selected from the group consisting of mis-localization of afarnesylated lamin, mis-localization of a non-farnesylated lamin,nuclear membrane disruption, aggregation of lamin, nuclear lobulation,nuclear blebbing, cytoskeleton disruption, early senescence, apoptosis,reduced secretion of MMP-3, or two or more thereof; and selecting acompound that alters the cellular defect so that it is more close tonormal.

Also provided is a method for identifying an agent useful for treating alaminopathy, which method involves contacting the agent to a cellexpressing progerin or a progerin-like abnormal lamin A protein underconditions sufficient to allow interaction between the cell and theagent; evaluating an amount of a cellular defect in the cell; andcomparing the amount of the cellular defect in the cell contacted withthe agent to an amount of the cellular defect in a control cell nottreated with the agent, wherein a statistically significant (e.g.,p<0.05, or p<0.01, or another selected level of significance)improvement in the amount of the cellular defect in the cell contactedwith the agent, as compared to the control cell not treated with theagent, identifies the agent as one that is useful for treating thelaminopathy.

Yet a further method provided herein is a method for identifying anagent that inhibits or reverses at least one cellular defect caused byconstitutive farnesylation of a lamin protein. Such a method involvescontacting a cell expressing progerin or a progerin-like abnormal laminA protein with a test agent under conditions sufficient to allowinteraction between the cell and the agent; and determining whether acellular defect caused by constitutive farnesylation of a lamin proteinis inhibited or reversed. By way of non-limiting example, the cellulardefect is mis-localization of a farnesylated lamin, mis-localization ofa non-farnesylated lamin, nuclear membrane disruption, aggregation oflamin, nuclear lobulation, nuclear blebbing, cytoskeleton disruption,early senescence, apoptosis, reduced secretion of MMP-3, or acombination of two or more thereof.

IV. Diseases Linked to Lamin A/C

Over 180 mutations have been reported in the LMNA gene, and currentlythere are eight diseases in addition to HGPS, referred to as the“laminopathies,” that are associated with various mutations in this gene(Gruenbaum et al., Nat. Rev. Mol. Cell. Biol. 6:21-31, 2005). Theseinclude such disorders as Emery-Dreifuss muscular dystrophy (EDMD; e.g.,associated with heterozygous R527P), mandibuloacral dysplasia(associated with homozygous R527H), atypical Werner's syndrome, dilatedcardiomyopathy-type 1A (CMD1A; e.g., associated with R644C), restrictivedermopathy, and Dunnigan-type familial partial lipodystrophy (Mounkes etal., Curr. Opin. Genet. Dev. 13:223-230, 2003; Navarro et al., Hum. Mol.Genet. 13:2493-2503, 2004).

Emery-Dreifuss muscular dystrophy is an autosomal recessive or dominantdisease characterized by muscle weakness, contractures, andcardiomyopathy with conduction defects. Familial partial lipodystrophy(Dunnigan variety) (FPLD) is an autosomal dominant disordercharacterized by marked loss of subcutaneous adipose tissue from theextremities and trunk but by excess fat deposition in the head and neck.This condition is frequently associated with profound insulinresistance, dyslipidemia, and diabetes.

Surprisingly, point mutations recently identified in the LMNA gene havebeen found to cause HGPS (PCT/US2003/033058, published as WO 04/035753,incorporated herein by reference in its entirety). The inheritance isnew mutation autosomal dominant, and identified mutations occur in codon608; the most common is due to a C to T base substitution in a CpGdinucleotide. It is currently believed that the mechanism of themutations is activation of a cryptic splice site within the LMNA gene,which leads to deletion of part of exon 11 and generation of a lamin Aprotein product that is 50 amino acids shorter than the normal protein.All of the identified mutations are predicted to affect lamin A but notLamin C. In addition, two cases of classical HGPS were identified withsegmental UPD (uniparental disomy) of chromosome 1q from fibroblast DNAdo not show the mutation, which may be indicative of a (in vivo or invitro) somatic rescue event.

The results described herein can be generalized to the aging process andrelated conditions and diseases, beyond progeroid diseases. This isbecause HGPS is in many respects closely connected to normal agingprocesses. HGPS continues to be recognized as a useful model of aging(Fossel, J. Pediatr Endocrinol Metab 13 Suppl 6:1477-1481, 2000). Forinstance, the connection to atherosclerosis is very strong, especiallyof the coronary arteries. In addition, alopecia in HGPS is similar tothat seen in subjects with advanced age. Further, the prime cellularfeature of HGPS, as described many years ago by Hayflick and others(Hayflick, N Engl J Med 295:1302-1308, 1976) is early cellularsenescence. The limited number of cell divisions in HGPS fibroblasts issimilar to what is seen in fibroblasts derived from elderly individuals.That was further explored by research showing similarities in the geneexpression patterns of HGPS fibroblasts and those derived from elderlypersons, distinguishing them from fibroblasts derived from youngerpersons (Ly et al., Science 287: 2486-2492, 2000).

V. Specific HGPS Mutations Identified in LMNA

The typical LMNA mutation in HGPS is a nucleotide substitution of a C toa T at position 1824 of SEQ ID NO: 19 (corresponding to position 4277 inGI 292250 (Accession No. L12401)), causing no change in the encodedamino acid (G608G) (Accession No. P02545), but creating a cryptic splicedonor site. Activation of this site results in an mRNA lacking 150nucleotides. This is in turn translated into a mutant protein with a 50amino acid internal deletion near the C-terminal end, termed progerin(Eriksson et al., Nature 423:293-298, 2003). Lamin A is normallyexpressed by most differentiated cells, where it integrally affects bothnuclear membrane structure and function (Rober et al., Development105:365-378, 1989). Progerin apparently acts in a dominant negativemanner on the nuclear function of cell types that express lamin A(Goldman et al., Proc. Natl. Acad. Sci. USA 101:8963-8968, 2004;Scaffidi & Misteli, Nat. Med. 11:440-445, 2005). In addition to thepotential mechanical fragility created by disrupting the nuclear lamina,this mutation also may affect other vital cellular processes such asgene transcription, DNA replication, and cell division.

There has also been identified a change at nucleotide position 4275 inGI 292250, which corresponds to amino acid 608 (corresponding toposition 1822 of SEQ ID NO: 19); this mutation changes a glycine to aserine in lamin A, and is predicted to generate the same cryptic splicesite as the silent mutation. Hence both mutations generate the samemutant lamin A protein (progerin). The two mutations both occur in thesame codon, which encodes amino acid 608.

The discovery that rare variants in the sequence of LMNA causes HGPSalso enables a variety of diagnostic, prognostic, and therapeuticmethods that are further embodiments. The new appreciation of the roleof lamin A in HGPS and more generally aging illnesses andarteriosclerosis/atherosclerosis enables detection of predisposition tothese conditions in a subject. This disclosure also enables earlydetection of subjects at high risk of these conditions, and providesopportunities for prevention and/or early treatment.

The deletion of the last half of exon 11 removes a cleavage site that isnormally necessary for processing of lamin A. Post-translationalmodification of the CAAX box (SEQ ID NO: 31) at the C-terminus of laminA, which is still present in the mutant forms, allows anchoring of theprotein in the membrane—but then this anchoring mechanism is normallyremoved by the processing cleavage. The lamin A mutant protein(progerin) shown herein is not cleaved, and thus appears to be trappedin this membrane location. Since lamin A is part of a large multiproteincomplex, its mislocalization is likely to pull other bystander proteinsinto the same improper location. It is now believed that this leads tostructural abnormalities of the nucleus that can be visualized by lightmicroscopy, immunohistochemistry, immunofluorescence, confocalmicroscopy, or electron microscopy. These characteristics are alsouseful for providing systems to test compounds for treatinglaminopathies.

VI. Farnesyltransferase Inhibitors to Ameliorate Progerin CellularDisruption

Farnesylation is a permanent posttranslational modification; therefore,proteolytic cleavage is required to remove the farnesylated residue fromlamin A protein and thereby effect defarnesylation and maturation offully processed lamin A. Although it can be farnesylated, the progerinprotein cannot be defarnesylated because it is missing the secondcleavage site necessary for binding the protease ZMPSTE24, whosecleavage activity removes the last 15 amino acids in the C-terminalportion of the protein and effects defarnesylation (see, for instance,Eriksson et al., Nature 423(6937):293-298, 2003, incorporated herein byreference). Progerin maintains its nuclear localization sequence and itsfarnesyl group, along with the central rod that allows dimerization withnormal lamin A and probably with itself. The result is an aberrantfarnesylated protein putatively capable of altering normal lamin Afunction as a dominant negative, as well as assuming its own aberrantfunction through its association with the nuclear membrane. Thus, themultisystem disease process and the variety of genes that are affectedin HGPS (Csoka et al., Aging Cell 3:235-243, 2004) lie downstream of aprotein defect that is central to basic cellular function.

The progerin deletion has a number of consequences which give rise tothe abnormal cellular phenotype, including abnormal protein processingand deletion of critical nuclear periphery protein binding domains. Theretention of the farnesyl group changes important properties of theprotein making it more lipophilic and altering interactions with otherproteins. We have demonstrated in vivo that progerin is incompletelyprocessed and retains the farnesyl group (Example 1). Expression of anN-terminal GFP-progerin fusion protein in normal fibroblasts causedabnormal nuclear morphology similar to that seen on HGPS fibroblasts.Furthermore, expression of the endogenous mutant in HGPS fibroblasts cancause abnormal localization of normal GFP tagged lamin A, demonstratinga dominant effect of the mutant protein on exogenously expressed normallamin A. However, expression of the normal GFP-lamin A did not improvethe nuclear morphology phenotype of HGPS fibroblasts, indicating thatthe deleterious effects of progerin are not compensated by increasedexpression of the normal protein.

It is also demonstrated herein that a protein cleavage site mutationconstruct gives rise to cellular phenotypes similar to a progerinconstruct (Example 2). A GFP-tagged cleavage-minus mutant lamin A(L647R) displayed a localization pattern very similar to thelocalization of GFP-tagged progerin when expressed in normalfibroblasts. Its expression caused a significant increase in abnormalnuclear morphology similar to the effect of the GFP-progerin construct.

Treatment of normal fibroblasts expressing progerin and HGPS fibroblastswith the farnesyltransferase inhibitor PD169541 resulted in aredistribution of GFP-lamin A and GFP-progerin localization andsignificant improvement in the nuclear morphology phenotype (Example 3).These studies indicate that abnormal farnesylation of progerin plays amajor role in the abnormal cellular phenotype and suggest a possibletherapeutic option for HGPS.

Based on the discoveries presented herein, it is shown thatfarnesyltransferase inhibitors (both direct effectors and indirectinhibitors) will inhibit or reduce the formation of progerin, cause adecrease in lamin A protein and an increase prelamin A protein.Decreasing the amount of aberrant protein may improve disease status inprogeria and other laminopathies; this is supported by the discoverythat treatment with an FTI reduces cellular defects linked to theexpression of progerin. It is therefore believed that afarnesyltransferase inhibitor, such as (but not limited to) PD169541,R115777 (tipifarnib, Zarnestra), and SCH66336 (lonafarnib, Sarasar),will reduce or abolish the formation of progerin, decrease lamin A, andincrease the levels of a prelamin A protein lacking 50 amino acids(“preprogerin”). This is demonstrated herein (Example 3), where normalfibroblasts expressing progerin and HGPS fibroblasts show aredistribution of GFP-lamin A and GFP-progerin localization andsignificant improvement in the nuclear morphology phenotype when exposedto the farnesyltransferase inhibitor PD169541.

Farnesyltransferase inhibitors can be used to reverse and/or preventcellular effects caused by accumulation of progerin or other forms ofconstitutively farnesylated lamin protein. This, it is now determinedthat FTIs can be used to treat effects associated with expression ofconstitutively farnesylated lamin A, including for instance symptomscharacteristic of HGPS, other laminopathies, aging-related conditions,atherosclerosis, and so forth. Specific cellular symptoms includemis-localization of farnesylated lamin, such a form of a lamin A protein(including prelamin A, progerin, preprogerin, and so forth),mis-localization of a non-farnesylated lamin, such as one or morenon-farnesylated lamin A protein form (for instance, fully processedlamin A protein that is mislocalized due to association with afarnesylated lamin protein form), aggregation of lamin(s), nuclearmembrane disruption, nuclear blebbing, nuclear lobulation, earlysenescence, apoptosis, and reduced secretion of MMP-3. In manyinstances, a cell will display a combination of two or more of thesesymptoms/effects.

VII. Farnesyltransferase Inhibitors

Farnesyltransferase inhibitors (FTIs) were developed originally asinhibitors of Ras biological activity, because FTase modification of Rasby farnesylation is essential for Ras to cause oncogenesis, and moregenerally as target-molecule specific chemotherapeutic agents. FTIsinhibit the posttranslational addition of a 15-carbon farnesyl group toa C-terminal cysteine residue that is required, for instance, forfarnesylated proteins (e.g., Ras) to localize to the cell membrane(Reuter et al., Blood 96(5):1655-1669, 2000). FTIs effectively blocksignaling and cellular transformation of some but not all isoforms ofRas, although it is certain that Ras is not the only target offarnesyltransferase inhibition (Ashar et al., J. Biol. Chem.275(39):30451-30457, 2000; Liu et al., Mol. Cell Biol. 20(16):6105-6113,2000). More generally, farnesyltransferase inhibitors were described aspotentially useful to treat cancers where mutated forms of Ras arecommonly found (e.g., colon and pancreatic cancers). However, since themost common forms of Ras found mutated in human cancers (K-Ras andN-Ras) undergo alternative prenylation by GGTase I when cells aretreated with FTI (Whyte et al., J. Biol. Chem. 272:14459-14464, 1997),the anti-tumor activity of FTIs is not attributed to blocking Rasitself. Alternatively prenylated Ras, for instance geranylgeranylatedK-Ras, retains function and is not inhibited by FTI. Instead, othersubstrates of FTase are believed to account for the clinical activity ofFTIs. Farnesylated proteins that become alternatively prenylated in thepresence of FTIs can escape the inhibitory effects of these compoundsand remain functional. Therefore, it is critical to evaluate thepossibility of alternative prenylation when determining whether aparticular protein, for example progerin, can be targeted successfullyby FTI treatment. Clinical consideration and prospects for FTIs ingeneral are discussed in Cox and Der (Curr. Op. Pharma. 2:388-393,2002).

FTIs generally can be divided into three groups: tetrapeptides having ormimicking the CAAX motif (SEQ ID NO: 31) (Brown et al., Proc. Natl.Acad. Sci. U.S.A. 89:8313-8316, 1992; Reiss et al., Proc. Natl. Acad.Sci. U.S.A. 88:732-736, 1991; Goldstein et al., J. Biol. Chem.266:15575-15578, 1991); analogs of farnesyl pyrophosphate (FPP) (Gibbset al., J. Biol. Chem. 268:7617-7620, 1993); and inhibitors withstructures not resembling either tetrapeptides or FPP (Liu et al., J.Antibiot. 45:454-457, 1992; Miura et al., FEBS Lett. 318:88-90, 1993;Omura et al., J. Antibiot. 46:222-228, 1993; Van Der Pyl et al. J.Antibiot. 45:1802-1805, 1992). The latter category of inhibitorsgenerally has lower activity compared to the first two categories. Byway of example, the FTI SCH66336 is a non-peptidomimetic FTI; FTI-277 isa peptidomimetic.

Several FTIs have entered into clinical trials in the last severalyears. For instance, the clinical candidate FTI SCH66336 (lonafarnib,Sarasar®) has been shown to inhibit proliferation of several humancancer cell lines and is active against human brain, lung, prostate,pancreas, colon, and bladder tumor xenografts in nude mice (Liu et al.,Cancer Res. 58(21):4947-4956, 1998; Feldkamp et al., Cancer Res.,61(11):4425-4431, 2001). One Phase I clinical trial showed that SCH66336inhibits protein farnesylation in vivo, and is generally well toleratedby the subject (Adjei et al., Cancer Res., 60(7):1871-1877, 2000).Anti-leukemic activity of SCH66336 on cell culture and mouse models ofBCR/ABL has been demonstrated (Reichert et al., Blood, 97(5):1399-1403;2001; Peters et al., Blood, 97(5):1404-1412, 2001).

The FTI R115777 (tipifarnib, Zarnestra®) has also entered clinicaltrials; it has been found to be well tolerated, but not fully effectiveagainst cancer (Adjei et al., J Clin Oncol. 21(9):1760-1766, 2003;Heymach et al., Annals of Oncology 15:1187-1193, 2004).

It is shown herein that FTIs can be used to reverse and/or preventcellular effects caused by accumulation of progerin or other forms ofconstitutively farnesylated lamin A. It is believed that all categoriesof FTIs may be used in methods and compositions provided herein; theselection of a specific FTI is within the skill of the ordinarypractitioner based on testing methods provided herein. In someembodiments, it is beneficial to select an inhibitor compound that ismore selective for farnesyltransferase, compared togeranylgeranyltransferase I. In other embodiments, it may be beneficialto select an inhibitor compound that is dually selective, in that itinhibits both FTase and GGTase I. Considerations for determiningselectivity criteria for FTIs include (but are not limited to) thepossibility of lower toxicity with FTase-specific FTIs versus dualspecificity FTIs, although both efficacy and toxicity may differaccording to the particular compound and the particular patient. As willbe recognized by an ordinarily skilled practitioner, otherconsiderations, for instance pharmacological and medical considerations,may also apply.

The development and chemistry of FTIs are well documented and known tothose of ordinary skill. By way of example, the following publicationsreview FTIs in the context of cancer treatment: Cox & Der, BiochimBiophys Acta 1333:F51-F71, 1997; Gelb et al., Curr Opin Chem Biol2:40-48, 1998; Rowinsky et al., J. Clin Oncol. 17; 3631-3652, 1999;Oliff, Biochim Biophys Acta 1423:C19-C30, 1999; Sebti & Hamilton, ExpertOpin Investig Drugs 9:2767-2782, 2000; and Gibbs et al., Curr Med Chem8:1437-1465, 2001.

The following patent documents provide additional descriptions of makingexample farnesyltransferase inhibitors and compositions contain suchcompounds: WO 00/39082; WO 00/12499; WO 00/12498; WO 00/01691; WO99/45912; WO 98/49157; WO 98/40383; WO 98/28303; WO 97/30992; WO97/21701; WO 97/16443; WO 94/10138; U.S. Application Publication20040063770; U.S. Application Publication 20030060450; U.S. Pat. No.6,187,786; U.S. Pat. No. 6,177,432; U.S. Pat. No. 6,169,096; U.S. Pat.No. 6,133,303; U.S. Pat. No. 6,037,350; U.S. Pat. No. 5,976,851; U.S.Pat. No. 5,972,984; U.S. Pat. No. 5,972,966; U.S. Pat. No. 5,968,965;U.S. Pat. No. 5,968,952; U.S. Pat. No. 5,965,578; U.S. Pat. No.5,965,539; U.S. Pat. No. 5,958,939; U.S. Pat. No. 5,939,557; U.S. Pat.No. 5,936,097; U.S. Pat. No. 5,891,889; U.S. Pat. No. 5,889,053; U.S.Pat. No. 5,880,140; U.S. Pat. No. 5,872,135; U.S. Pat. No. 5,869,682;U.S. Pat. No. 5,861,529; U.S. Pat. No. 5,859,015; U.S. Pat. No.5,856,439; U.S. Pat. No. 5,856,326; U.S. Pat. No. 5,852,010; U.S. Pat.No. 5,843,941; U.S. Pat. No. 5,807,852; U.S. Pat. No. 5,780,492; U.S.Pat. No. 5,773,455; U.S. Pat. No. 5,767,274; U.S. Pat. No. 5,756,528;U.S. Pat. No. 5,750,567; U.S. Pat. No. 5,721,236; U.S. Pat. No.5,700,806; U.S. Pat. No. 5,661,161; U.S. Pat. No. 5,602,098; U.S. Pat.No. 5,585,359; U.S. Pat. No. 5,578,629; U.S. Pat. No. 5,534,537; U.S.Pat. No. 5,532,359; U.S. Pat. No. 5,523,430; U.S. Pat. No. 5,504,212;U.S. Pat. No. 5,491,164; U.S. Pat. No. 5,420,245; and U.S. Pat. No.5,238,922.

Also useful are pharmaceutically acceptable acid or base addition saltsof FTIs, including any of the inhibitors as mentioned above. The phrase“pharmaceutically acceptable acid or base addition salts” includestherapeutically active non-toxic acid and non-toxic base addition saltforms which FTIs are able to form. Such compounds which have basicproperties can be converted in their pharmaceutically acceptable acidaddition salts by treating said base form with an appropriate acid.Appropriate acids comprise, for example, inorganic acids such ashydrohalic acids, e.g. hydrochloric or hydrobromic acid; sulfuric;nitric; phosphoric and the like acids; or organic acids such as, forexample, acetic, propanoic, hydroxyacetic, lactic, pyruvic, oxalic,malonic, succinic (i.e. butanedioic acid), maleic, fumaric, malic,tartaric, citric, methanesulfonic, ethanesulfonic, benzenesulfonic,p-toluenesulfonic, cyclamic, salicylic, p-aminosalicylic, pamoic and thelike acids.

FTI compounds which have acidic properties may be converted in theirpharmaceutically acceptable base addition salts by treating said acidform with a suitable organic or inorganic base. Appropriate base saltforms comprise, for example, the ammonium salts, the alkali and earthalkaline metal salts, e.g. the lithium, sodium, potassium, magnesium,calcium salts and the like, salts with organic bases, e.g. thebenzathine, N-methyl-D-glucamine, hydrabamine salts, and salts withamino acids such as, for example, arginine, lysine and the like.

The terms acid or base addition salt also comprise the hydrates and thesolvent addition forms that an FTI is able to form. Examples of suchforms are, for instance, hydrates, alcoholates and the like.

Also contemplated for use in methods and compositions described hereinare stereochemically isomeric forms of FTIs. The term stereochemicallyisomeric form includes all possible compounds made up of the same atomsbonded by the same sequence of bonds, but having differentthree-dimensional structures that are not interchangeable. Unlessotherwise mentioned or indicated, the chemical designation of a compoundencompasses the mixture of all possible stereochemically isomeric formsthat the compound may possess. Such mixture may contain alldiastereomers and/or enantiomers of the basic molecular structure of thecompound. Also contemplated are all stereochemically isomeric forms inpure form or in admixture with each other. Also contemplated aretautomeric forms of FTI compounds.

VIII. Methods of Treatment

The present disclosure includes methods of using various compounds andcompositions, including any of the listed FTIs or other compoundsidentified as capable of inhibiting, directly or indirectly,farnesylation of lamin A or progerin (referred to generally as FTIcompounds), as a treatment for disease. In a particular embodiment, theFTI compound is a peptidomimetic or non-peptidomimetic inhibitor of thefarnesyltransferase enzyme, and the treatment is a treatment of alaminopathy, such as HGPS or another progeroid disease or condition, anage-related condition, a cardiovascular disease or condition (such asatherosclerosis), and so forth.

The method includes administering a pharmaceutical agent or FTI, or morethan one, or a combination of a FTI (or more than one) and one or moreother pharmaceutical agents, to the subject in a pharmaceuticallycompatible carrier and in an amount effective to inhibit the developmentor progression of a disease. Although the treatment can be usedprophylactically in any patient in a demographic group at significantrisk for such diseases, subjects can also be selected using morespecific criteria, such as a definitive diagnosis of thedisease/condition or identification of one or more factors that increasethe likelihood of developing such disease (e.g., a genetic factor).

Various delivery systems are known and can be used to administerchemical compounds and FTIs as therapeutics. Such systems include, forexample, encapsulation in liposomes, microparticles, microcapsules,recombinant cells capable of expressing therapeutic molecule(s) (see,e.g., Wu et al., J. Biol. Chem. 262, 4429, 1987), construction of atherapeutic nucleic acid as part of a retroviral or other vector, andthe like. Methods of introduction include, but are not limited to,intrathecal, intradermal, intramuscular, intraperitoneal (ip),intravenous (iv), subcutaneous, intranasal, epidural, and oral routes.The therapeutics may be administered by any convenient route, including,for example, infusion or bolus injection, topical, absorption throughepithelial or mucocutaneous linings (e.g., oral mucosa, rectal andintestinal mucosa, and the like) ophthalmic, nasal, and transdermal, andmay be administered together with other biologically active agents.Pulmonary administration can also be employed (e.g., by an inhaler ornebulizer), for instance using a formulation containing an aerosolizingagent.

In a specific embodiment, it may be desirable to administer thepharmaceutical compositions of the invention locally to the area in needof treatment. This may be achieved, for example, and not by way oflimitation, by local infusion or perfusion (e.g., during surgery),topical application (e.g., in a dressing), injection, catheter,suppository, or implant (e.g., implants formed from porous, non-porous,or gelatinous materials, including membranes, such as sialasticmembranes or fibers), and the like. In one embodiment, administrationcan be by direct injection at the site (or former site) of a tissue thatis to be treated. In another embodiment, the therapeutic are deliveredin a vesicle, in particular liposomes (see, e.g., Langer, Science 249,1527, 1990; Treat et al., in Liposomes in the Therapy of InfectiousDisease and Cancer, Lopez-Berestein and Fidler (eds.), Liss, N.Y., pp.353-365, 1989).

In yet another embodiment, the therapeutic can be delivered in acontrolled release system. In one embodiment, a pump may be used (see,e.g., Langer Science 249, 1527, 1990; Sefton Crit. Rev. Biomed. Eng. 14,201, 1987; Buchwald et al., Surgery 88, 507, 1980; Saudek et al., N.Engl. J. Med. 321, 574, 1989). In another embodiment, polymericmaterials can be used (see, e.g., Ranger et al., Macromol. Sci. Rev.Macromol. Chem. 23, 61, 1983; Levy et al., Science 228, 190, 1985;During et al., Ann. Neurol. 25, 351, 1989; Howard et al., J. Neurosurg.71, 105, 1989). Other controlled release systems, such as thosediscussed in the review by Langer (Science 249, 1527 1990), can also beused.

The vehicle in which the agent is delivered can include pharmaceuticallyacceptable compositions of the compounds, using methods well known tothose with skill in the art. For instance, in some embodiments, chemicalcompounds, small molecules, and/or specifically FTIs typically arecontained in a pharmaceutically acceptable carrier. The term“pharmaceutically acceptable” means approved by a regulatory agency ofthe federal or a state government or listed in the U.S. Pharmacopoeia orother generally recognized pharmacopoeia for use in animals, and, moreparticularly, in humans. The term “carrier” refers to a diluent,adjuvant, excipient, or vehicle with which the therapeutic isadministered. Such pharmaceutical carriers can be sterile liquids, suchas water and oils, including those of petroleum, animal, vegetable, orsynthetic origin, such as peanut oil, soybean oil, mineral oil, sesameoil, and the like. Water is a preferred carrier when the pharmaceuticalcomposition is administered intravenously. Saline solutions, bloodplasma medium, aqueous dextrose, and glycerol solutions can also beemployed as liquid carriers, particularly for injectable solutions. Themedium may also contain conventional pharmaceutical adjunct materialssuch as, for example, pharmaceutically acceptable salts to adjust theosmotic pressure, lipid carriers such as cyclodextrins, proteins such asserum albumin, hydrophilic agents such as methyl cellulose, detergents,buffers, preservatives and the like.

Examples of pharmaceutical excipients include starch, glucose, lactose,sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate,glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol,propylene, glycol, water, ethanol, and the like. The therapeutic, ifdesired, can also contain minor amounts of wetting or emulsifyingagents, or pH buffering agents. These therapeutics can take the form ofsolutions, suspensions, emulsion, tablets, pills, capsules, powders,sustained-release formulations, and the like. The therapeutic can beformulated as a suppository, with traditional binders and carriers suchas triglycerides. Oral formulation can include standard carriers such aspharmaceutical grades of mannitol, lactose, starch, magnesium stearate,sodium saccharine, cellulose, magnesium carbonate, and the like. A morecomplete explanation of parenteral pharmaceutical carriers can be foundin Remington: The Science and Practice of Pharmacy (19th Edition, 1995)in chapter 95.

Embodiments of other pharmaceutical compositions are prepared withconventional pharmaceutically acceptable counter-ions, as would be knownto those of skill in the art.

Therapeutic preparations will contain a therapeutically effective amountof at least one active ingredient, preferably in purified form, togetherwith a suitable amount of carrier so as to provide proper administrationto the patient. The formulation should suit the mode of administration.

The composition of this disclosure can be formulated in accordance withroutine procedures as a pharmaceutical composition adapted forintravenous administration to human beings. Typically, compositions forintravenous administration are solutions in sterile isotonic aqueousbuffer. Where necessary, the composition may also include a solubilizingagent and a local anesthetic such as lignocaine to ease pain at the siteof the injection.

The ingredients in various embodiments are supplied either separately ormixed together in unit dosage form, for example, in solid, semi-solidand liquid dosage forms such as tablets, pills, powders, liquidsolutions, or suspensions, or as a dry lyophilized powder or water freeconcentrate in a hermetically sealed container such as an ampoule orsachette indicating the quantity of active agent. Where the compositionis to be administered by infusion, it can be dispensed with an infusionbottle containing sterile pharmaceutical grade water or saline. Wherethe composition is administered by injection, an ampoule of sterilewater or saline can be provided so that the ingredients may be mixedprior to administration.

The amount of the therapeutic that will be effective depends on thenature of the disorder or condition to be treated, as well as the stageof the disorder or condition. Effective amounts can be determined bystandard clinical techniques. The precise dose to be employed in theformulation will also depend on the route of administration, and shouldbe decided according to the judgment of the health care practitioner andeach patient's circumstances. An example of such a dosage range is 0.1to 200 mg/kg body weight in single or divided doses. Another example ofa dosage range is 1.0 to 100 mg/kg body weight in single or divideddoses.

The specific dose level and frequency of dosage for any particularsubject may be varied and will depend upon a variety of factors,including the activity of the specific compound, the metabolic stabilityand length of action of that compound, the age, body weight, generalhealth, sex, diet, mode and time of administration, rate of excretion,drug combination, and severity of the condition of the host undergoingtherapy.

The therapeutic compounds and compositions of the present disclosure canbe administered at about the same dose throughout a treatment period, inan escalating dose regimen, or in a loading-dose regime (e.g., in whichthe loading dose is about two to five times the maintenance dose). Insome embodiments, the dose is varied during the course of a treatmentbased on the condition of the subject being treated, the severity of thedisease or condition, the apparent response to the therapy, and/or otherfactors as judged by one of ordinary skill in the art. In someembodiments long-term treatment with the drug is contemplated.

In some embodiments, sustained localized release of the pharmaceuticalpreparation that comprises a therapeutically effective amount of atherapeutic compound or composition may be beneficial. Slow-releaseformulations are known to those of ordinary skill in the art. By way ofexample, polymers such as bis(p-carboxyphenoxy)propane-sebacic-acid orlecithin suspensions may be used to provide sustained localized release.

It is specifically contemplated in some embodiments that delivery is viaan injected and/or implanted drug depot, for instance comprisingmulti-vesicular liposomes such as in DEPOFOAM® drug delivery agent(SkyePharma, Inc, San Diego, Calif.) (see, for instance, Chamberlain etal., Arch. Neuro. 50:261-264, 1993; Katri et al., J. Pharm. Sci.87:1341-1346, 1998; Ye et al., J. Control Release 64:155-166, 2000; andHowell, Cancer J. 7:219-227, 2001).

IX. Cell-Based Systems for Identifying and Characterizing FTIs forTreatment

Also provided herein are in vivo assay systems designed to show theability of test compounds (test agents) to inhibit farnesyltransferaseand more specifically, ability of test compounds to reduce or ameliorateone or more cellular effects caused by constitutive farnesylation oflamin A.

If farnesylation is inhibited, maturation of lamin A cannot be completedand the precursor (prelamin A) accumulates. This phenomenon has beenrecognized (see, e.g., Beck et al., J. Cell Biol. 110:1489-1499, 1990;Kilic et al., J. Biol. Chem. 272:5298-5304, 1997). Thus, inhibition offarnesylation can be detected by detecting increased accumulation ofprelamin A (e.g., on a gel based on differential mobility, or using anantibody that preferentially detects prelamin A). One method forgenerating a prelamin A-specific antibody preparation is described inAdjei et al. (Clin Cancer Res 6:2318-2325, 2000). Detection of prelaminA is recommended as a marker of farnesyltransferase inhibition in thatsame reference. It was shown to be detectable using Western blotting andimmunohistochemical staining, as well as differential migration onSDS-PAGE.

Similarly, inhibition of farnesyltransferase can be measured bydetecting changes in processing, and therefore changes inelectrophoretic mobility, of other proteins. By way of example, thechaperone HDJ-2 [Neckers et al., The Hsp90 chaperone family, In: D. S.Latchmann (ed.), Stress Proteins, pp. 9-42 (New York: sprinter-Verlag,1999)] and the peroxisomal protein Pxf (James et al., J. Biol. Chem.269:14182-14190, 1994) are good candidate markers for monitoringfarnesyltransferase inhibition (Adjei et al., Clin Cancer Res6:2318-2325, 2000).

In addition, specific examples provided herein describe constructsuseful in cell-based systems. These systems can be used, for instance,in studying the effectiveness of specific FTIs (or other candidatetherapeutic compounds) to ameliorate symptoms (particularly, cellulardefects) of constitutive farnesylation of lamin protein forms. Specificsystems therefore exploit methods of detecting and analyzingmis-localization of a farnesylated lamin, mis-localization of anon-farnesylated lamin, nuclear membrane disruption, aggregation oflamin protein, nuclear lobulations, nuclear blebbing, cytoskeletondisruption, early senescence, apoptosis (e.g., through measurement ofAnnexin V), reduced secretion of MMP-3, and so forth. It is contemplatedthat certain of such cell-based systems could be used in ahigh-throughput format, in order to more rapidly analyze the effect andeffectiveness of known and putative inhibitory molecules.

Examples of described constructs, cell-based systems, and transgenicanimals are specifically contemplated for use in characterizing,testing, selecting, and identifying compounds that may be putativefarnesyltransferase inhibitory.

The following examples are provided to illustrate certain particularfeatures and/or embodiments. These examples should not be construed tolimit the invention to the particular features or embodiments described.

EXAMPLES Example 1 Incomplete Lamin A Processing in HGPS Leads toNuclear Abnormalities

This example describes methods for detecting lamin A in cells, andexamining cellular characteristics that result from expressing progerin.

GFP-tagged normal and mutant LMNA expression constructs have beencreated, and normal and HGPS fibroblasts have been transfected withthese constructs in order to examine protein localization and effect onnuclear structure. Expression of the mutant lamin A protein (progerin)in normal fibroblasts caused abnormal nuclear morphology includingmislocalization of progerin to foci at the nuclear periphery and nuclearblebbing similar to that seen in HGPS fibroblasts. Expression of normallamin A in HGPS fibroblasts did not significantly correct the nuclearblebbing phenotype. Furthermore, the normal GFP-lamin A wasmislocalized, indicating that the presence of the endogenous mutant canact in a dominant-negative fashion. The progerin deletion could have anumber of consequences which could give rise to this abnormal cellularphenotype, including abnormal protein processing and deletion ofcritical protein binding domains. The deleted region includes a proteincleavage site that normally removes 15 amino acids including a CAAX box(SEQ ID NO: 31) farnesylation site.

Methods

Constructs: Normal and mutant alleles of lamin A/C were cloned byisolating RNA from the cell lines AG11513a and NF, respectively. Anoligo dT primer was used for first strand synthesis using theSUPERSCRIPT® reverse transcriptase kit (Invitrogen). The first strandcDNA was used as a template to amplify normal and mutant products usingthe Platinum Taq high fidelity kit from Invitrogen with the primerslaminAF1 (SEQ ID NO: 1) and laminAR1 (SEQ ID NO: 2). Due to the GC-richnature of the template, DMSO and betaine were added to finalconcentrations of 4% and 500 mM, respectively. The reaction was run for20 cycles with an annealing temperature of 62° C. and the resultingproduct was used for TOPO TA cloning (Invitrogen). These cDNA constructswere verified by sequencing and used as templates for subcloning intothe pEGFP-C2 and pRevTRE vectors (Clontech).

Cell Culture: Cell lines used were normal fibroblast lines, GM08398 andHGMDFN090, and HGPS fibroblast lines: P01 (HGADFN003), HGADFN004,HGADFN136, AG03513d, AG10750 and AG11498. Fibroblasts were cultured inminimal essential medium (Gibco, #10370-021) supplemented with 15% FBS(HiClone), 2 mM L-glutamine, penicillin (50 U/ml) and streptomycin (50mg/ml). The Amphotropic Phoenix retroviral packaging line (Peter Nolan,Stamford University) was maintained in DMEM supplemented with 10% FBS, 2mM L-glutamine, penicillin (50 U/ml) and streptomycin (50 mg/ml).

Retroviral Constructs: The pRevTetOff vector and pRevTRE constructs weretransfected using FUGENE®6 transfection reagent into the AmphotropicPhoenix retroviral packaging line (Peter Nolan, Stamford University).Forty-eight hours after transfection the media containing retrovirus wasfiltered through a 0.22 micron filter and used immediately or stored at−70° C. Retroviral infection was performed by exposing target cells to a1:1 mix of retroviral supernatant and growth medium with 4 μg/mlpolybrene.

GFP localization: Normal and HGPS fibroblasts were plated on coverslipsin six well plates or in chambered microscope slides (BD Biosciences) 24hours prior to transfection. Fibroblasts were transiently transfectedwith the GFP-lamin A or GFP-progerin expression constructs usingFUGENE®6 transfection reagent (Roche). Seventy-two hours later the cellswere washed two times with PBS (pH 7.2) and fixed for 20 minutes at 4°C. with 4% paraformaldehyde in PBS containing 62.5 g/ml LPC palmitoyland 1 U/ml rhodamine phalloidin (Molecular Probes) to stain thecytoskeleton. Following three washes with PBS, Prolong Gold mountingmedium with DAPI (Molecular Probes) was applied to the coverslips whichwere then inverted and placed on microscope slides.

Immunocytochemistry: Immunocytochemistry (ICC) was performed aspreviously described. Cells were cultured on coverslips in 6-well platesfor 72 hours. Coverslips were washed twice with PBS and fixed with 4%paraformaldehyde in PBS with 0.18% Triton X-100 for 20 min at 4° C.Coverslips were washed 3 times with PBS and incubated 30 min at 37° C.in blocking buffer (1% BSA in PBS). The cells were probed with 1:10dilution of the lamin A specific antibody (USBiological) in blockingbuffer for 1 hour at room temperature followed by washing three timeswith PBS. Secondary antibody (Goat Anti-Mouse IgG, FITC) was diluted1:1000 in blocking buffer and applied to the coverslips for 1 hour atroom temperature. After washing three times for 5 min with PBS, thecoverslips were inverted and placed on slides with ProLong Gold mountingmedium containing DAPI (Molecular Probes).

Immunoprecipitation: Cells from nearly confluent T75 flasks wereharvested and lysed in RIPA buffer containing a protease inhibitorcocktail (Roche). Cell pellets were vortexed and sonicated briefly toensure thorough lysing. 500 mg of total cellular protein in a finalvolume of 500 μl from each sample was immunoprecipitated as follows.Cell lysate was precleared by incubating for 1 hour with protein Gsepharose at 4° C. on a nutator mixer. The protein G sepharose wasremoved by a brief spin and the supernatant was incubated with 100 μlanti-lamin A/C antibody (Abcam ab8984) overnight at 4° C. Fifty μlprotein G Sepharose was added to each sample and incubated for 3 hour at4° C. After washing four times with RIPA buffer, 50 μl 2× NuPAGE samplebuffer with reducing agent (Invitrogen) was added to the protein Gpellet, heated to 100° C. for 10 min, and loaded onto a 4-12% Bis-TrisNuPAGE gel (Invitrogen).

Immunoblotting: Proteins were transferred to Immobilon-P membrane(Millipore) using a Trans-Blot semi-dry electrophoretic transfer cell(BioRad) blocked with 5% dry milk in TBST and probed with lamin A/Cspecific antibodies according to standard protocols.

Results GFP Localization

When GFP-tagged normal lamin A was expressed in normal fibroblasts, thenuclei had an oval shape with fluorescence evenly distributed at theperiphery of the nucleus (FIGS. 4A and 4B). Expression of the GFP-taggedprogerin in normal fibroblasts caused abnormal nuclear morphology,similar to that seen in HGPS fibroblasts including nuclear blebbing, andthe mislocalization of the GFP-tagged progerin into nuclear foci in manycells (FIG. 4C). Expression of normal lamin A in HGPS fibroblasts for 72hours did not significantly correct the nuclear blebbing phenotype seenin these cells, and the normal GFP-lamin A was mislocalized, indicatingthat the presence of the endogenous mutant can cause mislocalization ofthe normal GFP-tagged lamin A. These experiments were repeated usingconfocal microscopy in order to verify these results and gain very highresolution images that better show the localization of GFP-progerin(FIG. 6). The most commonly seen localization pattern for theGFP-progerin 72 hours post-transfection appears as thick regions in thenuclear lamina. These were also detected by ICC in HGPS fibroblasts withless resolution most likely due to a lack of availability of the epitopewithin these aggregates (FIG. 5).

GFP-lamin A and GFP-progerin were also expressed from tet-off retroviralconstructs that allow for sustained expression and modulation ofexpression to address the possibility of results due to over-expressionof the fusion proteins. Reducing expression of GFP-progerin (as measuredby Western blotting) did not significantly alter the localization of themutant protein into aggregates at the nuclear periphery.

Progerin Processing

In order to determine if the progerin protein retains a farnesyl groupan immunoprecipitation (IP) was performed using anti lamin A/Cantibodies (Abcam ab8984). The IP step was necessary because as many as0.1% of cell proteins are modified by the addition of farnesyl orgeranylgeranyl group. Progerin was efficiently detected following IP asa protein of approximately 74 kDa that runs between lamin C (70 kDa) andlamin A (78.5 kDa) on an immunoblot, consistent with the loss of 50amino acids from lamin A (FIG. 7C). The presence of a ³H-labeledfarnesyl group on the progerin protein was detected by fluorography ofthe immunoprecipitated proteins. A farnesyl group was not detected onnormal lamin A so the normal protein was completely processed in thesecells. The addition of 2 μM FTI prevented the farnesylation of progerinand lamin A. In addition, the pre-lamin A band in these lanes was shownby the presence of a larger band since farnesylation was necessary forthe subsequent processing steps including removal of the C-terminal 18amino acids.

Discussion

To date, there have been seven diseases associated with mutations in theLMNA gene. The disorders include muscular dystrophy, lipodystrophy andneuropathy, and the tissues most affected in these disorders arestriated muscle, adipose tissue and bone (reviewed in Burke & Stewart,Nat Rev Mol Cell Biol 3:575-585, 2002). The remarkable heterogeneity andphenotypic differences of the laminopathies are reflected in thedistribution of mutations in LMNA. Virtually all HGPS patients have thesame mutation creating an abnormal splice donor site in exon 11 of theLMNA gene. The mis-splicing creates a protein missing 50 amino acids ina globular domain near the C-terminus. Mutations causing Dreifussmuscular dystrophy (EDMD2) are distributed throughout the gene, whilethose resulting in familial dilated cardiomyopathy type 1 (CMD1A) aremainly clustered within the coiled-coil domain, suggesting that thelatter may act by disrupting dimerization or subsequent polymerizationof lamin A/C. In contrast, the mutations found in individuals with HGPS,Dunnigan familial partial lipodystrophy (FPLD), and mandibuloacraldysplasia (MADA or MAD) are found within exons encoding the C-terminalglobular domain of lamin A/C and, more specifically, are restricted tojust a few codons in exons 8, 9 and 11 (Eriksson et al., Nature423:293-298, 2003; Shackleton et al., Nat Genet. 24:153-156, 2000;Speckman et al., Am J Hum Genet. 66:1192-1198, 2000; Novelli et al., AmJ Hum Genet. 71:426-431, 2002). Abnormal nuclear morphology is seen inmany of the laminopathies, including Emory (EDMD2) and FPLD, so it seemsunlikely that this cellular phenotype alone accounts for theHGPS-specific disease phenotypes.

We and others have described the mis-splicing caused by the activationof a cryptic splice site due to the codon 608 mutation in HGPS. Themis-splicing results in loss of 150 nucleotides between the crypticsplice site and the end of exon 11. The frame of the transcript isretained so that the final nine codons from exon 12 are transcribedincluding a CAAX box (SEQ ID NO: 31) farnesylation site. However, thedeletion of 50 amino acids may have numerous consequences, includingdeletion of protein binding domains and deletion of the cleavage sitenecessary for normal protein processing and predicted removal of thefarnesyl group from the mature protein. Incomplete processing of lamin Aappears to be an important factor as mutations in ZMPSTE24 (the proteasethat performs the final cleavage step removing the farnesyl group) arefound in patients with a severe form of mandibuloacral dysplasia.Although the conserved cleavage site is missing in progerin, it remainedpossible that the protein was still cleaved as there are potential sitesin the progerin protein that may have substituted for the missingcleavage sequence. Thus, while maintenance of the farnesyl group ispredicted, it had not been formally proven.

Data presented in this example show that the progerin protein remainsfarnesylated and is not cleaved. The retention of the farnesyl group mayhave numerous consequences. Farnesyl groups increase lipophilicity andare involved in membrane association. Also, farnesyl groups are involvedin protein interactions. In fact, the protein Narf has been shown tointeract only with the farnesylated form of lamin A (Barton & Worman, JBiol Chem. 274:30008-30018, 1999). Retention of the farnesyl group onthe progerin protein and on the cleavage minus mutant alters thelocalization of these proteins in comparison the normal lamin A. Thisabnormal distribution of the nuclear lamin A may contribute to the lossof integrity of the lamina and thus result in the abnormal nuclearphenotype observed in HGPS cells and when these proteins are expressedin normal cells.

N-terminal GFP-lamin A fusions have been shown previously to displaylocalization patterns consistent with localization of the non-taggedprotein. The fusion protein has the added advantage of showing laminstructures that are difficult to detect using ICC because epitopes arenot available.

Example 2 Cleavage Mutant of Lamin A

As shown in Example 1, the C-terminus of progerin was not cleaved andthe protein remained farnesylated. This example provides theconstruction of a cleavage-site mutation in lamin A, and compares itsprocessing and cellular effects with those of progerin. Transfection ofnormal cells with the codon 647 cleavage site mutation construct wasfound to give rise to similar nuclear abnormalities as those seen inprogeria cells. These results indicate that abnormal farnesylation ofprogerin plays a major role in the abnormal cellular phenotype.

Methods Creation of Cleavage Minus Mutant

The L647R mutant was introduced into the pRevTRE-GFP-lamin A constructusing a PCR-based site directed mutagenesis. Two overlapping PCRproducts were generated using the following primer pairs: PCR product 1using the primers LMNA-1320-For (SEQ ID NO: 3) and LMNA-L647R-Rev (SEQID NO: 4), and PCR product 2 using the primers LMNA-L647R-For (SEQ IDNO: 5) and pRevTRE-3700-Rev (SEQ ID NO: 6). The reverse primer forproduct 1 is the complement sequence to the forward primer for product 2and both of these primers contain a single mismatch with the targetsequence that will generate the L647R mutation.

Both products were gel purified and used together as the template for athird PCR reaction using the primers LMNA-1390-For (SEQ ID NO: 7) andpRevTRE-3440-Rev (SEQ ID NO: 8). Although the template is twooverlapping fragments, a single PCR product results, which was gelpurified and cloned using the TOPO-TA kit (Invitrogen). The resultinginsert was subcloned into the pRevTRE-GFP-lamin A construct using Mfe Iand Cla I restriction endonucleases. The resulting construct,pRevTRE-GFP-LA(L647R) was sequenced to verify the presence of theintroduced mutation without any random base changes due to the use ofPCR generate the construct.

Results and Discussion

A mutant lamin A was engineered to create a L647R amino acid change inthe cleavage site of lamin A, so that the effect of retaining thefarnesyl group could be assessed without the 50 amino acid deletion thatoccurs in the progerin protein. The L647R mutation changed a conservedbase in the cleavage site of lamin A so that ZMPSTE24 could not cleavethe protein, leaving it farnesylated.

A GFP-tagged cleavage-minus mutant lamin A (L647R) displayed alocalization pattern nearly identical to the localization of GFP-taggedprogerin when expressed in normal fibroblasts (FIG. 8). Its expressioncaused a significant increase in abnormal nuclear morphology, similar tothe effect of the GFP-progerin construct (Example 1) (FIG. 9). Thisresult demonstrates that incomplete processing of lamin A such that itretains the farnesyl group can cause morphological changes in cellssimilar to those caused by progerin expression.

Example 3 Farnesyltransferase Inhibitor Affects HGPS NuclearAbnormalities

This example demonstrates that treatment of cells expressing progerinwith a farnesyltransferase inhibitor reverses cellular morphologydefects. The FTI used in this example, PD169451, was a gift from Pfizer.

Effect of FTI, PD169451, on Transiently Expressed GFP-Lamin A andGFP-Progerin

Normal and HGPS fibroblasts were plated on coverslips in 6 well plates24 hours prior to transfection. Normal fibroblasts were transientlytransfected with the GFP-lamin A or GFP-progerin expression constructsusing FUGENE®6 transfection reagent (Roche). The following day, themedium was replaced with growth medium containing 0 nM, 100 nM, 500 nMor 1 mM of the FTI, PD169451. Forty-eight hours later the cells werewashed two times with PBS (pH 7.2) and fixed for 20 min at 4° C. with 4%paraformaldehyde in PBS containing 62.5 μg/ml LPC palmitoyl and 1 U/mlrhodamine phalloidin (Molecular Probes) to stain the cytoskeleton.Following three washes with PBS, 10 μl of ProLong Gold mounting mediumwith DAPI (Molecular Probes) was applied and the coverslips, which wereinverted onto a glass microscope slide.

Effect of FTI, PD169451, on the Nuclear Morphology of Normal and HGPSFibroblasts

Normal and HGPS fibroblasts were maintained in T75 tissue culture flaskswith 0 nM, 10 nM, and 100 nM concentrations of the FTI, PD169451, addedto the growth medium. The medium was changed every 2 days and the cellswere split when they were approximately 80% confluent. Three days priorto each time point, a flask was trypsinized and cells were placed inchambered microscope slides (BD Biosciences) and maintained in theappropriate FTI concentration. Seventy-two hours later the cells werewashed two times with PBS (pH 7.2) and fixed for 20 minutes at 4° C.with 4% paraformaldehyde in PBS containing 62.5 g/ml LPC palmitoyl and 1U/ml rhodamine phalloidin (Molecular Probes) to stain the cytoskeleton.Following three washes with PBS, 10 l of ProLong Gold mounting mediumwith DAPI (Molecular Probes) was applied and the cells were overlaidwith a coverslip.

Results and Discussion

The effects of several concentrations of the specificfarnesyltransferase inhibitor Pfizer PD 169451 on nuclear morphology andlocalization of lamin A and progerin were examined. Exposure of 100 nMFTI for 72 hours had a significant effect on the localization oftransiently expressed GFP-lamin A and GFP-progerin (FIGS. 7A and 7Brespectively). Lamin A and progerin were localized in very similarfashion to intranuclear filaments when exposed to the FTI rather thanlocalizing to the nuclear periphery (compare FIGS. 7A and 7B with FIGS.6A and 6B). In addition, short-term exposure to the FTI had asignificant effect on the nuclear morphology of fibroblasts expressingGFP-progerin. These fibroblasts had a 33% decrease in the percentage ofcells with abnormal nuclear morphology (FIG. 7D). To verify this resultin HGPS fibroblasts, the AG11498 line was exposed to various FTIconcentrations for three days. Treating these HGPS cells resulted in asmuch as a 40% drop in the percent of cells with abnormal nuclearmorphology (FIG. 7E).

A longer duration analysis on the effects of FTI on the nuclearmorphology of normal and HGPS fibroblasts revealed that a sustained lowdose of 10 nM substantially reduced the percentage of cells displayingabnormal nuclear morphology (FIG. 10). High doses of the FTI, greaterthan 200 nM, caused cells to stop dividing after a week to 10 days, andcaused an increase in abnormal nuclear morphology in normal fibroblasts.However, 10 nM FTI did not substantially alter the nuclear morphology ofnormal cells and significantly lowered the percentage of abnormal nucleiin HGPS fibroblasts and in normal fibroblasts expressing GFP-progerin.Four HGPS cell lines were maintained in growth media containing 10 nM or100 nM FTI. After two days exposure to 10 nM FTI the four HGPS celllines demonstrated approximately a 40% reduction in the percent of cellswith abnormal nuclei. At six days, the HGPS lines displayed a 72%, 70%,45%, and 27% reduction in abnormal nuclei after six days of 10 nM FTI.After 14 days, three HGPS lines each had at least a 70% decrease inabnormal nuclei and one HGPS cell line (HGADFN004) had a 12% drop inabnormal morphology. This cell line failed to grow at the 14 day timepoint when exposed to 100 nM FTI. Exposure to the FTI causes someabnormal nuclear morphology in normal fibroblasts in a dose dependentmanner.

Because the progerin protein maintains the farnesyl group, FTIs mightimprove the nuclear morphology of HGPS fibroblasts and normalfibroblasts expressing progerin by sequestering the mutant protein intothe intranuclear filaments. These results showed a significant reductionin the abnormal nuclear phenotype in cells treated with an FTI. The FTIused in these experiments, PD169451, is an isoprene competitor which isspecific for farnesyltransferase and does not inhibit the relatedgeranylgeranyl transferase I. FTIs have been investigated as anticanceragents because farnesylation of Ras is necessary for Ras signaling andbiological activity. Other FTIs have been used in clinical studies andare well tolerated by patients. The improvement in the morphology of theHGPS cells when exposed to low doses of the FTI indicates that theseagents may be useful in treating the disease. The effect of these drugsin animal models of HGPS may be analyzed to determine if there isimprovement in the overall phenotype of the animals. The adverse effectsof higher concentrations of the FTI on nuclear morphology and cellgrowth and the reduced effect on a higher passage HGPS line may indicatethat there is a therapeutic window for effective concentrations of thedrug and time of treatment for the patient.

Example 4 Effects of FTI Treatment on Expression of L647R CleavageMutant

The effects of exposure to the FTI PD 169451 on cells expressing thelamin A L647R cleavage mutant are being tested. Based on the resultsobserved with progerin (Example 3), it is believed that the FTI willcause the GFP-tagged L647R mutant to localize to intranuclear filaments,as observed when GFP-lamin A and GFP-progerin are expressed in cellsexposed to the FTI. This result is expected because lamin A (and itsmutant forms) must be farnesylated in order to be localized to thenuclear lamina. The FTI causes these forms of lamin A to accumulate inthe interior of the nucleus. It is also predicted that the increase inabnormal nuclear morphology observed when the GFP-L647R mutant isexpressed in normal fibroblasts will be alleviated by FTI treatment, asobserved herein for normal fibroblasts expressing GFP-progerin whentreated with the FTI.

By way of example, GFP-lamin A and GFP-progerin were expressed fromtet-off retroviral constructs that allowed for sustained expression andmodulation of expression to address the possibility of results due toover-expression of the fusion proteins when the proteins weretransiently expressed. In addition, a cleavage minus mutant,GFP-LA(L647R), was included to examine the effect of retaining thefarnesyl-group without the 50 amino acid deletion found in progerin.

When no tetracycline was present, the GFP fusions were expressed atrelatively high levels as measured by Western blot (FIG. 11 D).

Addition of 0.05 mg/ml tetracycline allowed for reduced expression ofthe GFP fusions such that near-endogenous levels were obtained, while 8mg/ml tetracycline reduced, but did not altogether prevent, expressionfrom these constructs (FIG. 11 D). When GFP-progerin was stablyexpressed in normal fibroblasts (0.05 mg/ml tetracycline) at levelssimilar to progerin levels in HGPS fibroblasts, 10 nM FTI substantiallyreduced the percent abnormal nuclei by 71% (p<0.0001) (FIG. 11B).

When GFP-tagged L647R mutant was expressed at intermediate levels (0.05mg/ml tetracycline), the increase in abnormal nuclear morphology wasreduced 67% by exposure to 10 nM FTI (p<0.0001) (FIG. 11 B). Theimprovement in abnormal nuclear morphology with intermediate expressionlevels of GFP-progerin and GFP-LA(L647R) was actually less profound atthe higher concentrations of the FTI (100 nM) in comparison to thedecrease observed for 10 nM (FIG. 11 B). Treatment with 10 nM FTI couldnot overcome the effects of very high expression levels of theGFP-progerin or GFP-LA(L647R) that were obtained when no tetracyclinewas present to repress expression (FIG. 11 A). However, the cleavageminus mutant may be more tractable to FTI treatment at the highestexpression level, especially when exposed to 100 nM FTI, perhaps due toslightly lower expression levels. Some leaky expression was observedeven at 8 mg/ml tetracycline, and these low levels did affect nuclearmorphology, causing a moderate increase in the percent abnormal nucleiin the GFP-progerin and GFP-LA(L647R) cells (FIG. 11 C). Thus, it isunlikely that the effects seen with the progerin constructs are duesolely to protein over expression.

As with transient expression, stable expression of GFP-lamin A did nothave a significant effect on nuclear morphology, and the GFP signal wasevenly distributed at the nuclear periphery (FIG. 12 A).

When GFP-progerin was expressed in normal fibroblasts at levels nearwhat is observed in HGPS patients, nuclear aggregates at the peripheryof the nucleus and intranuclear structures were observed, along with asubstantial increase in abnormal nuclear morphology including foldingand blebbing of the nuclear membrane (FIG. 12 D). In addition, chromatinstaining by DAPI was altered with a loss of staining often observed atthe nuclear periphery and in regions adjacent to the progerinaggregates. Expression of intermediate levels of the cleavage minusmutant, GFP-LA(L647R), had very similar effects on localization of theGFP signal and on nuclear morphology as the GFP-progerin (FIG. 12 G).Also, as observed in the transient expression studies, the FTI causedthe GFP-lamin A and GFP-progerin to localize to intranuclear filamentswhen expressed in cells exposed to the FTI at 100 nM concentrations.Localization of GFP-tagged L647R mutant was also redistributed tointranuclear filaments when exposed to 100 nM FTI (FIG. 12 I). However,10 nM FTI did not lead to this drastic reorganization of GFP signal forany of the fusion proteins. Rather, alterations in GFP localization atthis FTI concentration were less noticeable (FIG. 12 B, E and H).

Material included in this example was also published as Glynn & Glover,Hum Mol Genet. 14: 2959-2569, 2005, Sep. 6, 2005, which is incorporatedherein by reference in its entirety.

Example 5 Testing FTIs in HGPS, Experimental Protocols

This example describes production of a series of expression constructsfor lamin A, progerin, and derivative proteins having mutations in theCAAX box (SEQ ID NO: 31). Methods are provided for analyzing suchexpression constructs in vivo. These methods are examples of analyseswhich can be used for determining the effectiveness offarnesyltransferase inhibitors (FTIs), potential FTIs, and other drugsand compounds, for reversing or reducing cellular defects caused byaccumulation of progerin or other farnesylated proteins.

Mutant Construction

A set of lamin A CAAX (SEQ ID NO: 31) mutants was generated, includingCSIM (SEQ ID NO: 32) to CSIL (SEQ ID NO: 34) and to SSIM (SEQ ID NO: 33)mutants in constructs expressing wildtype or del150 lamin A proteins(Map 1 of FIG. 11). These expression constructs were made according toprotocol using the QUIKCHANGE®II XL Site-Directed Mutagenesis Kit(Stratagene, LaJolla, Calif.). Primers were designed using QUIKCHANGE®site directed mutagenesis Primer Design (Stratagene), synthesized andpurified by polyacrylamide gel electrophoresis at Integrated DNATechnologies, Inc. (Coralville, Iowa).

Primer sequences for the cDNA constructs were as follows: SSIM_Sense(SEQ ID NO: 11); SSIM_Anti-sense (SEQ ID NO: 12); CSIL_Sense (SEQ ID NO:13); and CSIL_Anti-sense (SEQ ID NO: 14). Primer sequences for theminigene constructs were as follows: SSIM_Sense (SEQ ID NO: 15);SSIM_Anti-sense (SEQ ID NO: 16); CSIL_Sense (SEQ ID NO: 17); andCSIL_Anti-sense (SEQ ID NO: 18).

The “del150” lamin A framework provides a protein that has deleted fromit the same 50-amino acids that are deleted in the HGPS protein. Map 1of FIG. 13 provides an overview of these constructs. Specific examplesof entire coding sequence (SEQ ID NOs: 19, 21, 23, 25, 27, and 29) forspecific lamin A proteins (SEQ ID NOs: 20, 22, 24, 26, 28, and 30) areshown herein, which correspond respectively to constructspEGFP_LA_myc_wildtype CSIM (the “normal” lamin A), pEGFP_LA_myc_wildtypeCSIL, pEGFP_LA_myc_wildtype SSIM, pEGFP_LA_myc_del150 CSIM,pEGFP_LA_myc_del150 CSIL, and pEGFP_LA_myc_del150 SSIM.

Cell Culture: For immunofluorescence, HeLa cells were plated in chamberslides at 25,000 cells per chamber and cultured in DMEM containing 10%FBS, penicillin-streptomycin, and L-glutamine until 90-95% confluentprior to transfection.

Transfection: The following constructs were transfected by lipofectioninto the HeLa cells using the LIPOFECTAMINE® 2000 transfection reagent(Invitrogen, Carlsbad, Calif.) transfection procedure for DNA:

-   -   pEGFP_LA_myc_del150 CSIL cDNA;    -   pEGFP_LA_myc_del150 SSIM cDNA;    -   pEGFP_LA_myc_wildtype CSIL cDNA;    -   pEGFP_LA_myc_wildtype SSIM cDNA;    -   pEGFP_LA_myc_wildtype CSIM cDNA (−control);    -   pEGFP_LA_myc_del150 CSIM cDNA (+control); and    -   mock transfection vector only (CMV driven EGFP vector with no        insert).

Optionally, for each transfection, Western analysis can be performed todetermine levels of progerin, prelamin, and lamin A. Using themyc-epitope tag engineered into these constructs, endogenous protein canbe differentiated from the protein produced from the transfectedconstruct.

Transfection efficiency was determined by the visual inspection of EGFPsignal versus the DAPI staining of nuclei. Transfection efficiency forcells on all slides was estimated to be 30-40%.

Fluorescence and Electron Microscopy: At different time points (e.g., 48and 72 hours after transfection), cells grown on coverslips were washedwith PBS and fixed with 3.7% formaldehyde in PBS for 10 minutes at roomtemperature. After fixation, cells were washed three times with PBS,then permeabilized with 0.5% Triton X-100 in PBS for 5 minutes at roomtemperature, then rinsed with PBS. Cells were then overlaid with primaryantibody, a rabbit anti-prelamin A from Dr. Michael Sinensky (1:5000 inPBS with 10% FBS; Cancer Research 54: 3229-3232; 1994), and incubated atroom temperature for 1-2 hours. After removal of the primary antibodies,samples were washed once with PBS and incubated for 30 minutes in thedark at room temperature with a mixture of affinity-purifiedrhodamine-conjugated goat anti-rabbit IgG (2 μg/ml; Alexa fluor 594(red); Molecular Probes, Eugene, Oreg.), washed three times with PBS,and dehydrated in increasing concentrations of ethanol baths (70, 90,100%) for two minutes per solution. 20 μl of a 50:50Vectashield:Vectashield with DAPI (Vector Laboratories, Burlingame,Calif.) mixture was placed on the center of each pad and a coverslip wasplaced on top. Slides were then examined on a Zeiss Axioplanfluorescence microscope. LMNA was viewed using natural EGFP expressionand prelamin A with the Sinensky Ab and Alexa Fluor594.

Results and Discussion

After 48 hours of incubation, the HeLa cells transfected with thepEGFP_LA_myc_wildtype CSIM cDNA construct (the negative control)appeared normal with the nuclear rim of transfected cells expressing thegreen EGFP marker for lamin A. These nuclei appeared to have normalovoid and elliptical morphologies by both the DAPI and EGFP staining. Incontrast, the positive control (the pEGFP_LA_myc_del150 CSIM cDNAprogerin construct) displayed dramatic nuclear blebbing and lobulationsby both DAPI and EGFP staining, identical to the blebbing seen in thenuclei of patients with HGPS.

The mutant pEGFP_LA_myc_wildtype SSIM cDNA construct demonstrated normalnuclear morphology, as would be expected. However, in this case, all ofthe EGFP staining appeared as aggregates within the nucleoplasm, with noEGFP at the nuclear rim, representing the new localization of the laminA when it is unable to be farnesylated and directed to the nuclearmembrane. Similarly, these nucleoplasmic aggregates were seen in themutant pEGFP_LA_myc_del150 SSIM cDNA progerin construct. In this casehowever, there was no nuclear blebbing or lobulations as would beexpected with the progerin construct.

The pEGFP_LA_myc_wildtype CSIL cDNA construct provided more of anintermediate picture. All the cells maintained their ovoid, ellipticalshape in terms of morphology. Lamin A localization as determined by EGFPexpression was a mix between many cells with normal nuclear membranestaining and many others with staining occurring only in thenucleoplasmic aggregates as seen in the SSIM mutants.

Likewise, the pEGFP_LA_myc_del150 CSIL cDNA construct also displayed amixed phenotype, with a moderate amount of blebbing occurring in somecells mixed with many completely normal looking cells. Similarly,approximately half of the cells appeared to have normal lamin A EGFPstaining at the nuclear rim, while the other half only expressed lamin Ain nucleoplasmic aggregates.

After incubating transfected cells for 72 hours, all of the aboveresults were unchanged except those concerning the CSIL mutants. In thecase of the pEGFP_LA_myc_wildtype CSIL cDNA construct, there was a muchgreater percentage of cells appearing to express lamin A only in thenucleoplasm, and far fewer cells appearing like normal wildtype cellswith lamin A only at the nuclear rim. The pEGFP_LA_myc_del150 CSIL cDNAprogerin construct also appeared more like the SSIM del150 mutant, withfar fewer blebbed cells and greater percentage of cells with normalovoid, elliptical shapes and lamin A EGFP expression localized only inthe nucleoplasm.

Example 6 Minigene Constructs for Lamin A Mutants

Additional constructs can be made, in which the intron between the LMNAexon 11 and 12 is included. A progerin protein can be produced from suchconstructs if a mutation that activates the cryptic splice site found inHGPS is included. These constructs are referred to as “minigene”constructs. An overview of a representative minigene construct is shownin Map 2 of FIG. 13.

The following constructs are made and used to transfect cells asdescribed in Example 5

-   -   pEGFP_LA_(—)75_myc_del150 CSIL minigene;    -   pEGFP_LA_(—)75_myc_del150 SSIM minigene;    -   pEGFP_LA_(—)77_myc_wt CSIL minigene;    -   pEGFP_LA_(—)77_myc_wt SSIM minigene;    -   pEGFP_LA_myc_wt CSIM minigene (−control);    -   pEGFP_LA_myc_del150 CSIM minigene (+control); and    -   mock transfection vector only (CMV driven EGFP vector with no        insert).

Cells transfected with minigene constructs are subjected to analysis asdescribed in Example 5. These minigene constructs will be useful todetermine if the efficiency of splicing may affect results such as thosenoted in Example 5.

Also contemplated are minigene constructs in which the expression of thelamin A/progerin/mutation protein is under inducible control. By way ofexample, tetracycline inducible constructs are contemplated (see, forinstance, Map 3 of FIG. 13).

Example 7 Treatment with FTIs

Constructs such as those described in Examples 5 and 6 can be used incell-based and animal expression systems to further characterize knownFTIs and potential farnesyltransferase inhibitory compounds, inparticular as regards the effectiveness of such compounds to amelioratecellular defects associated with constitutive farnesylation of lamin A.By way of example, FTIs are reconstituted in DMSO and stored at −20° C.until used. The following concentrations of two specific FTIs will beused to determine their ability to prevent or reduce nuclear blebbing inthe pEGFP_myc_del150 (CSIM) progerin construct and pEGFP_myc_wt (CSIM)LMNA construct:

R115777 SCH66336   50 nM (.05 μM)   50 nM (.05 μM)  100 nM (.1 μM)  100nM (.1 μM)  200 nM (.2 μM)  200 nM (.2 μM)  500 nM (.5 μM)  500 nM (.5μM)  1000 nM (1 μM)  1000 nM (1 μM)  2000 nM (2 μM)  2000 nM (2 μM) 5000 nM (5 μM)  5000 nM (5 μM) 10000 nM (10 μM) 10000 nM (10 μM)

Cells are cultured in media containing one of the indicated levels ofFTI for 4-5 days or until 90-95% confluent prior to transfection.Transfected cell samples are examined by Western blot analysis todetermine endogenous and transfected levels of progerin, prelamin, andlamin A.

Morphometric Analysis

To determine the ability of FTIs to prevent nuclear blebbing, 200 nucleifrom both treated and untreated cell cultures transfected with thepEGFP_myc_del150 (CSIM) progerin construct or the pEGFP_myc_wt (CSIM)LMNA construct are scored either as lobulated, if they contain more thantwo lobulations, or not lobulated. These determinations are made byusing a double-blind approach, followed by averaging the two data sets.Degree of farnesylation inhibition is determined by the percentage ofcells demonstrating the presence of prelamin A.

Contour analysis as in Goldman et al. (Proc. Natl. Acad. Sci. U.S.A.8963-8968, 2004) can be used to accurately quantitate nuclear morphologyirregularities. To analyze the changes in nuclear shape, the perimeterand the area of nuclei is measured in cells at different passages. Thismeasurement will be carried out on the midsection of the nucleus byusing the overlay/measure function in the LSM software (Zeiss). Todetermine the extent of nuclear lobulation, 50-200 randomly selectednuclei are measured per passage and the nuclear roundness or contourratio (4×area/perimeter²) is calculated. The contour ratio for a circleis one. As the nucleus becomes more lobulated, this ratio approacheszero. To calculate the perimeter length and area, the outline of nucleiis traced with a closed loop drawing tool using images captured fromcells prepared for immunofluorescence with LA Ab (Anti-lamin A/CMonoclonal Antibody, Unconjugated, Clone JoL2, MAB3211; Chemicon,Temecula, Calif.). Lamin fluorescence that is associated with the edgeof the lobulations and deep invaginations that typify the surface oflater-passage HGPS cells will be included. Significance of the resultantobservations can be measured using, for instance, a two-tailed Student ttest.

Example 8 Animals Engineered to Express Lamin A/Progerin or MutantsThereof

Mutant or transgenic organisms that under-express or over-express laminA protein are useful for research and characterization, for instance,the characterization of FTIs and potential inhibitory molecules. Theyalso allow insight into the physiological and/or pathological role oflamin A in a healthy and/or pathological organism, for instance incharacterization of aging and aging-related diseases and conditions,including progeria and atherosclerosis. These mutants are “geneticallyengineered,” meaning that information in the form of nucleotides hasbeen transferred into the mutant's genome at a location, or in acombination, in which it would not normally exist. Nucleotidestransferred in this way are said to be “non-native.” For example, anon-LMNA promoter inserted upstream of a native LMNA encoding sequencewould be non-native. An extra copy of an LMNA gene on a plasmid,transformed into a cell, would be non-native.

Mutants may be, for example, produced from mammals, such as mice, thateither over-express lamin A or under-express lamin A, or that do notexpress lamin A at all, or that express a mutant form of lamin A (suchas the splice variant described herein), that express one or more ofthese proteins under control of an inducible promoter, and so forth.Over-expression mutants can be made by increasing the number of LMNAgenes in the organism, or by introducing an LMNA gene into the organismunder the control of a constitutive or inducible or viral promoter suchas the mouse mammary tumor virus (MMTV) promoter or the whey acidicprotein (WAP) promoter or the metallothionein promoter. Mutants thatunder-express lamin A may be made by using an inducible or repressiblepromoter, or by deleting the LMNA gene, or by destroying or limiting thefunction of the LMNA gene, for instance by disrupting the gene bytransposon insertion.

A gene is “functionally deleted” when genetic engineering has been usedto negate or reduce gene expression to negligible levels. When a mutantis referred to in this application as having the LMNA gene altered orfunctionally deleted, this refers to the LMNA gene and to any orthologof this gene. When a mutant is referred to as having “more than thenormal copy number” of a gene, this means that it has more than theusual number of genes found in the wild-type organism, e.g., in thediploid mouse or human.

A mutant mouse or other animal over-expressing normal or mutant lamin Amay be made by constructing a plasmid having an LMNA encoding sequencedriven by a promoter, such as the mouse mammary tumor virus (MMTV)promoter or the whey acidic protein (WAP) promoter. This plasmid may beintroduced into mouse oocytes by microinjection. The oocytes areimplanted into pseudopregnant females, and the litters are assayed forinsertion of the transgene. Multiple strains containing the transgeneare then available for study.

WAP is quite specific for mammary gland expression during lactation, andMMTV is expressed in a variety of tissues including mammary gland,salivary gland and lymphoid tissues. Many other promoters might be usedto achieve various patterns of expression, e.g., the metallothioneinpromoter.

An inducible system may be created in which the subject expressionconstruct is driven by a promoter regulated by an agent that can be fedto the mouse, such as tetracycline. Such techniques are well known inthe art.

In particular, one example transgenic animal is a mouse model of HGPS,duplicating one of the G608G mutations. The mouse sequence is perfectlyidentical at this region of the coding sequence, so this would producethe same kind of consequence for lamin A as in the human.

In addition to knock-out systems, it is also beneficial to generate“knock-ins” that have lost expression of the wildtype protein but havegained expression of a different, usually mutant form of the sameprotein.

By way of example, the dominant mutant lamin A protein (progerin)provided herein, or any of the other mutant lamin A, progerin, orprogerin-like proteins described herein, can be expressed in a knockoutbackground, such as a mutant mouse that has been rendered defective orselectively defective (e.g., inducibly knocked-out) for LMNA expression,in order to provide model systems for studying the effects of treatmentwith FTIs or potential farnesyltransferase inhibitory compounds. Inparticular embodiments, the resultant knock-in organisms provide systemsfor studying aging, arteriosclerosis, and/or HGPS-like conditions.

Those of ordinary skill in the relevant art know methods of producingknock-in organisms. See, for instance, Rane et al. (Mol. Cell. Biol.,22: 644-656, 2002); Sotillo et al. (EMBO J., 20: 6637-6647, 2001); Luoet al. (Oncogene, 20: 320-328, 2001); Tomasson et al. (Blood, 93:1707-1714, 1999); Voncken et al. (Blood, 86: 4603-4611, 1995); Andrae etal. (Mech. Dev., 107: 181-185, 2001); Reinertsen et al. (Gene Expr., 6:301-314, 1997); Huang et al. (Mol. Med., 5: 129-137, 1999) by way ofexample.

Example 9 Mouse Systems for Testing FTIs

Mice from positive progerin-expressing lines, or lines expressing otherabnormal lamin proteins, can be treated with differing doses of FTIs andcompared with controls for the prevention or amelioration of anyphenotypic changes (for instance, growth changes, ability to reproduce,hair loss, skin changes, vascular changes, and nuclear blebbing).

One specific dosing regimen for examining FTI effects in transgenic miceis as follows, administered (e.g., orally, such as by oral gavage) overdiffering time periods, for instance 10 days to 3 weeks:

R115777 SCH66336 10 mg/kg BID 10 mg/kg BID 25 mg/kg BID 25 mg/kg BID 50mg/kg BID 50 mg/kg BID 100 mg/kg BID  100 mg/kg BID Alternatively, an FTI can be provided in food. By way of example, an FTI(such as FTI R115777) can be provided in by way of animal (e.g., mouse)feed at doses as follows: 450 mg/kg of feed, 150 mg/kg of feed, and 0mg/kg of feed (as a negative control). One of ordinary skill willrecognize that all of these dosages are examples only, and can bevaried.

Example 10 Vascular Disease Process in Hutchinson-Gilford ProgeriaSyndrome

HGPS skin fibroblasts displayed disruption of nuclear architecture,premature senescence, apoptosis, decreased hyaluronan secretion anddecreased matrix metalloproteinase secretion. The disruption of nucleararchitecture and apoptosis has been examined in vascular smooth musclecells (VSMC) transfected with progerin. In addition, by transfecting theprogeria minigene into normal VSMC and endothelial cells (EC), it wasshown that these cell types produce progerin. It was hypothesized thatdisruption of nuclear structure and function culminates in prematurecell death and cell senescence of VSMC and EC. It is believed that thesefeatures can be improved or reversed by blocking post-translationalprocessing of lamin A using farnesyltransferase inhibition, and thatelimination of progerin will restore normal function to progeria andother laminopathy cells.

LMNA is a 12 exon gene that initially produces a pre-lamin A which mustbe processed both outside of the nucleus via isoprenylation of a carboxyterminal CAAX motif (SEQ ID NO: 31), cleavage of the last three aminoacids, and methyl esterification, and inside of the nucleus viaproteolysis of its terminal 18 amino acids to become mature lamin A(Sinensky et al., J Cell Sci 107(Pt 1):61-7, 1994). The deletion in HGPSprobably does not affect the ability of Progerin to localize to thenucleus or to dimerize (as normal lamin A does), because the componentsneeded for nuclear localization and dimerization are not deleted. Theability to isolate progerin from cells (FIG. 15), yet detect lamins onlyin the nucleus via anti-lamin A using immunofluorescence (FIG. 19),supports this hypothesis.

A. Senescence and Apoptosis

Many of the features of HGPS, such as atherosclerosis and bony changes,may be at least partially attributed to premature cellular senescence orapoptosis in vivo. The data demonstrate that HGPS fibroblasts have lessthan half of the growth potential and show premature apoptosis ascompared to normal skin fibroblasts from age-matched or older donors.These investigations have been extended to HGPS-transformed vascularcell types and senescence and apoptosis have been addressed in theprincipal cell types involved in abnormalities of vasculature (VSMC andEC).

Cell Culture: VSMC and EC were kindly provided by Richard Karas, MD,PhD, New England Medical Center, Boston, Mass. HGPS and normalfibroblasts cells were obtained from the Coriell Cell Repositories andthe Progeria Research Foundation.

Dermal fibroblasts were maintained in MEM with Earl's Salts (Gibco,10370-021) (EMEM), 1×Pen./Strep. (100 U/ml P, 100 μg/ml S), 2 mML-Glutamine and 15% fetal bovine serum, under 5% CO₂. Cell numbers wereobtained at every passage using a hemocytometer and Trypan Blue cellviability staining. VEC were grown on gelatin (Sigma #G1393) in Medium199 in Earl BSS with 2 mM L-glutamine, 0.05-0.1 mg/mL Endothelial CellGrowth Supplement (Fisher #CB40006B)*, 0.10 mg/ml Heparin (Sigma H3149),1×Pen/Strep, and 10% fetal bovine serum. VSMC were grown with eitherDMEM+10% Fetal Bovine Serum+2 mM L-glutamine (1×) and 1×Pen/Strep (asabove) or Clonetics® Media Systems SmGM2 media (CC-3182).

Cell cultures were fed every 2-3 days, and split when necessary using0.05% trypsin.

Assay for growth rate: Growth rate was assessed by measuring populationdoubling times. Population doublings (PD)=log₂(N/N₀), where N=final cellnumber and N₀=starting cell number. Therefore, PD=(log₁₀ N/N₀)/0.301 andPD time=Total incubation time/PD.

Senescence-associated β-galactosidase (SA-β gal) and cell cycling:Pre-confluent cultured cells were fixed with 3% formaldehyde and stainedwith 1 mg/ml 5-bromo-4-chloro-3-indolyl β-D-galactoside (X-gal), 40 mMcitric acid/sodium phosphate buffer pH 6.0, 5 mM potassium ferrocyanide,5 mM ferricyanide, 150 mM sodium chloride and 2 mM magnesium chloride.At pH 6.0 the solution specifically stains senescent cells (cells inG1-like arrest) blue in the perinuclear region (Dimri et al., Proc NatlAcad Sci U S A. 92:9363-93637, 1995). Exposure to 10 μCi/ml-³H-thymidinefollowed by fixation and development for silver grains was used to assaycell cycling.

Results and Discussion

In vitro, fibroblasts normally progress to senescence, and this isconsidered a hallmark of “aging” in vitro. Fibroblasts from older donorssenesce more quickly than those from younger donors (Martin et al.,Laboratory Investigation 23:86-92, 1970). There are conflicting reportson the growth potential of HGPS fibroblasts (Goldstein, J Inv. Derm.73:19-23, 1979). Seven different HGPS skin fibroblast lines have beencultured, and they have senesced completely, or have reached Phase 3growth, as indicated by an increase in doubling time and changes inmorphology (Hayflick, J Am Geriatr Soc 22(1):1-12, 1974), betweenpassages 6 and 18 (Table 1). This is less than half of the growthpotential of normal skin fibroblasts from age-matched or older donors(Schneider & Mitsui, Proc. Nat. Acad. Sci. 73:3584-3588, 1976) (FIG.17). In general, the senescing HGPS cells are increased in size and showcharacteristic intracellular changes associated with senescence, whereasnormal fibroblasts after similar passage number do not. Another hallmarkof senescence is cytoplasmic staining with SA-β gal (Dimri et al., ProcNatl Acad Sci USA. 92:9363-93637, 1995). A higher percentage of HGPSfibroblasts stained positively for SA-β gal activity than normalfibroblasts of similar passage, while the same normal fibroblastsincorporated a much higher percentage of ³H-thymidine than the HGPSfibroblasts, indicating increased cell cycling.

TABLE 1* Percentages of cycling and senescent cells Normal Control HGPS% % PD Passage SA-β % PD time Passage SA-β % time # gal Thymidine (h) #gal Thymidine (h) 12 6 80 44.8 9 11 67 132 10 20 90 58.9 13 56 22 236 160 85 54.5 16 48 15 431 *each line represents a separate experiment; 200cells randomly assayed

B: Nuclear Morphology and Disease Phenotype

Immunofluorescent Staining for Lamin A and Nuclear Morphology: Cellswere seeded into glass coverslips, methanol fixed, incubated in primary(mouse anti-human lamin A/C monoclonal used at 1:10, MAB3211, Chemicon)and fluorescent secondary (Alexa Fluor 488 goat anti-mouse IgG used at1:3000, A-11001, Molecular Probes) antibodies and counter-stained withDAPI. Confocal Microscopy can also be used to better identify nuclearblebbing.

Assays for apoptosis: The occurrence of apoptosis in normal versusHGPS-transformed cultures was determined using an Annexin V bindingassay (Vybrant® Apoptosis Assay Kit #2, Molecular Probes) adapted foradherent cell cultures. Alternatively, caspase analysis may be used forapoptosis analysis, using known protocols.

Results and Discussion

Alteration of lamin A produces blebbing of nuclei in cultured HGPSfibroblasts (Eriksson et al., Nature 423(6937):293-298, 2003) and thedata show that this nuclear blebbing increases significantly withpassage number. Early passage, vigorously growing, normal fibroblastswith low levels of senescence associated β-galactosidase (SA-β gal)staining (6%; FIG. 17, Point A) had normal, round nuclei (FIG. 19A), ablebbing rate of 12% (Table 2), and virtually no apoptotic cells (FIG.18). This rate increased to 22% blebbing at passage 12. Early passage,vigorously growing, HGPS fibroblasts with correspondingly low levels ofSA-β gal staining (6%; FIG. 17, Point C) demonstrated abnormally blebbednuclei in 11% of cells and this percentage grew to 28% at passage 12 andthen 50% at passage 14, when growth curves demonstrated almost completesenescence in the HGPS fibroblasts (Table 2).

TABLE 2 Percentage of nuclei exhibiting abnormal morphology (blebbing).passage # cells Condition number counted # blebs % blebs Normal p9 11314 12.4% Normal p12 102 22 21.5% HGPS htert p28 106 17 16.0% HGPS htertp34 111 26 21.6% HGPS p6 120 13 10.8% HGPS p12 125 35 28.0% HGPS p14 15075 50.0%

The percentage of cells with nuclear blebbing increased with passagenumber in HGPS fibroblasts (Table 2). The potential causal associationof nuclear blebbing and thus nuclear membrane instability withsenescence and apoptosis in HGPS-transformed vascular cells is beingstudied further. To understand whether blebbing precedes or coincideswith these events, cultures are co-stained for senescence or apoptosisand for nuclear morphology at varying cell passages.

C: ECM and Vascular Disease in HGPS

There is considerable indication, both in vivo and in vitro, that HGPSis a disease heavily involving abnormalities in the extracellularmatrix, with increased collagen and elastin secretion, disorganizeddermal collagen, and decreased decorin over normal controls (reviewed inDavidson et al., Ciba-Found-Symp. 192:81-99, 1995). Extracellular matrixmolecules have both structural and cell signaling functions in skin,bone, and the cardiovascular system all severely affected in HGPS.

Reduction in hyaluronic acid (HA)-dependent pericellular matrices (PCM)(HA-PCM) may be involved in prevention of cellular senescence by actingas a protective sink for reactive oxygen species. Reactive oxygenspecies are produced as a normal consequence of the aerobic cellularmetabolism. However, oxidative stress induces senescence and apoptosisand is implicated in etiology of a long list of diseases, including HGPS(Yan et al., Biochemical and Biophysical Research Commun. 257:163-167,1999) and atherosclerosis (reviewed in Napoli et al., J. Cell. Biochem.82:674-682, 2001), as well as the process of normal aging (Floyd et al.,Exp. Gerontol. 36:619-640, 2001). Abnormalities in the extracellularmatrix (ECM) may be key components to disease process in HGPS.Hyaluronan-cell interactions promote cell growth and survival signalingpathways. Published microarray analyses (Csoka et al., Aging Cell3:235-43, 2004) of HGPS fibroblasts and our protein data show dramaticdecreases in stromelysin (MMP-3) over normal control fibroblasts.

MMP's are key components of ECM restructuring for angiogenesis. Bymeasuring the size of HA-PCM and MMP-3 in VSMC and EC, a correlationbetween premature cellular senescence and decreased matrix assembly inHGPS cultures can be substantiated.

HA-PCM assay: HA-PCM size was analyzed using a well-established particleexclusion assay (Knudson & Toole, Dev Biol 112(2):308-318, 1985).Briefly, a suspension of formalin-fixed erythrocytes (0.8 ml of 10⁸cells/ml in 0.1% BSA in PBS) was added to a 35 mm dish of sub-confluentfibroblasts, settled, and photographed using a 4.3 megapixel digitalcamera (VersaCam Digital Imaging Outfit, Chestnut Hill, Mass.).Morphometric analysis of the HA-PCMs was determined using Scion ImageBeta 4.0.2 software (Scion Corp., Frederick, Md.) for analysis of cellperimeter and matrix perimeter.

MMP-3 Analysis: MMP-3 can be assayed from confluent culture conditionedmedia using a standard ELISA kit (Amersham MMP3 ELISA kit (RPN2613)).

Results and Discussion

Hyaluronan production by normal and HGPS fibroblasts was measured.Hyaluronan content in pre-senescent HGPS and age-matched normal controlfibroblasts was analyzed using fluorophore-associated carbohydrateelectrophoresis analysis, revealing a 70% decrease in both the celllayer and culture medium over age matched control cultures.

TABLE 3 Hyaluronan Content in Fibroblast Cultures (ng/ml)* Cell LayerMedia Layer Normal Pre-senescent 1005 572 HGPS Senescent 136 343 *ageand passage - matched (n = 2); ELISA-like assay

Dermal fibroblasts, VSMC and EC exhibit highly hydrated HA-PCMs that canbe visualized indirectly by their ability to exclude particles (seemethods section). Preliminary data suggested that the HA-PCM thicknessof senescent HGPS fibroblasts was 12-fold decreased over age matchedpre-senescent HGPS fibroblasts and 22-fold decreased over normal dermalfibroblasts (Table 4).

TABLE 4 Mean HA-PCM radius in cultured fibroblasts (μm)* NormalPre-senescent 6.7 Normal Senescent 1.5 Progeria Pre-senescent 3.7Progeria Senescent 0.3 *1 × 10⁴ cells were plated per 35 mm dish, andHA-PCM radii were measured on 25 cells randomly chosen prior to additionof fixed red blood cells.

The data substantiate a correlation between premature cellularsenescence and decreased matrix assembly in HGPS fibroblast cultures.Large polymeric hyaluronan protects against oxidative stress byabsorbing free radicals and inhibiting pro-inflammatory cytokineformation (reviewed in Neumann et al., FEBS Letters 453:283-7, 1999).This evidence suggests that changes in hyaluronan (previously namedhyaluronic acid or hyaluronate) precede onset of standard senescentcharacteristics in HGPS fibroblasts but resemble changes that also occurduring senescence of normal fibroblasts. These results are of broadinterest because hyaluronan is involved in the development ofatherosclerotic lesions and has also been found to play a role in cellsurvival signaling cascades.

Matrix Metalloproteinase-3 (MMP-3) has been associated with cellularsenescence (Parrinello et al., J. Cell Sci. 118:485-496, 2005),atherosclerotic plaque formation (Doherty et al., Mayo Clin. Sci.79:197-210, 2004), and HGPS (Csoka et al., Aging Cell 3:235-243, 2004;Ly et al., Science 287:2486-2492, 2000). A complete absence of MMP-3 wasobserved in HGPS dermal fibroblast cultures, as compared to normalcontrol dermal fibroblast cultures (FIG. 20). This molecule thereforeserves as a biomarker of cellular disease in HGPS, which would benormalized with effective treatment of disease process, and helps toelucidate the mechanism of disease in HGPS.

D: Vascular Changes and HGPS

Atherosclerosis in HGPS is a diffuse process, involving the largevessels, the coronary arteries, and distal vasculature. To date therehave been fewer than 20 autopsies described on children with HGPS(reviewed in Stehbens et al., Cardiovasc Pathol 10(3):133-136, 2001).These studies note focal atherosclerotic plaques throughout large andsmall arteries, including all coronary artery branches, interstitialfibrosis and stenosis. Plaques are markedly calcified with cholesterolcrystals evident. Microscopy reveals thickened and almost acellularhyaline fibrosis. Notably, two autopsies report a paucity of medialsmooth muscle cells, with acellular fibrous tissue predominating in themedia. Intimal and medial collagen have been noted as disorganized andof small diameter. However, a major problem with studying autopsyspecimens is that we are observing the end stage of a complicatedprocess (Zhang et al., Am J Pathol. 143:496-506, 1993).

Calcification of both vascular intima and media are early events in thedevelopment of atherosclerotic plaques in aging individuals and thosewith diabetes (Cooper et al., Am J Hypertens. 14:475-86, 2001).Apoptosis occurs in vascular smooth muscle cells (VSMC) prior to thedevelopment of calcification (Proudfoot et al., Circulation 106:3044-50,2002), and may even be required for calcification to occur. Vascularcalcification is implicated in both pathological and in vitro studies asa requisite event in plaque formation. Therefore apoptosis may be a keyelement to development of disease in HGPS. Premature apoptosis occurs inHGPS fibroblasts (FIG. 18).

E: Creating HGPS in Vascular Cells:

One way to begin understanding the initial phases of atherosclerosis inHGPS is to study the VSMC and EC at initial phases of disease. This canbe achieved by introducing the HGPS gene, or constructs expressingsimilar mutant forms of lamin A, into normal VSMC and EC. If progericVSMCs have limited capacity for proliferation, as do fibroblasts (FIG.15), then the clonal and proliferative requirements of VSMC in plaqueinitiation may lead to early senescence or apoptosis of VSMC andeventually result in the lack of VSMC seen at autopsy.

Constructs were created that allowed VSMC and EC to demonstrate naturalsplicing capabilities, thus demonstrating that these cell types doexpress progerin in vivo.

Recombinant DNA constructs using site directed mutagenesis: Briefly,RT-PCR products of LMNA were generated using RNA from normal dermalfibroblasts and the GC-rich protocol for SUPERSCRIPT® reversetranscriptase First-Strand Synthesis System for RT-PCR (Invitrogen) withPlatinum Taq High Fidelity and PCRx Enhancer System (Invitrogen). TheLMNA cDNA was then cloned using a pCR2.1-TOPO cloning vector(Invitrogen) and One Shot TOP10 Chemically Competent E. coli(Invitrogen).

Normal Lamin A: The normal lamin A cDNA was cloned via RT-PCR from RNAextracted from normal human dermal fibroblast line GM00969D (CoriellRepositories). The cDNA was inserted into the pEGFP-C mammalianexpression vector (Clontech) at the EcoRI site (FIG. 14).

Progerin: The mutant HGPS splice variant of lamin A was created from thenormal lamin A construct via site directed mutagenesis (Stratagene).Bases 1818 through 1968 were deleted from exon 11 producing a cDNA thatcodes for the mutant protein “progerin” with a GFP fused to its Nterminus (FIG. 14).

Minigene: A third construct was created, which contained the intronbetween exon 11 and 12 as well as the c-to-t point mutation in codon 608in exon 11 (G608G) responsible for HGPS. The presence of this intronallows a cell expressing the construct to recognize the cryptic splicesite caused by the point mutation, and splice both the normal andabnormal lamin A products (FIG. 14).

Quantitative PCR: Total RNA was extracted from cells harvested viatrypsinization using the RNEASY® Mini RNA isolation Kit (Qiagen). Fivehundred nanograms (500 ng) of each mRNA template was reverse-transcribedwith an oligo (dT) primer using the SUPERSCRIPT II® reversetranscriptase first strand cDNA synthesis kit (Invitrogen). QuantitativePCR was then carried out on a Stratagene MX 4000 apparatus using theQUANTITECT® SYBR Green PCR analysis kit from Qiagen with the followingprimers: Forward (SEQ ID NO: 9) and Reverse (SEQ ID NO: 10).

Vascular cells (EC, FIG. 16A; coronary artery SMC, FIG. 16B; radialartery SMC, FIG. 16C) were electroporated and exposed to either aprogerin minigene or LMNA-lamin A minigene. These cell types weredemonstrated to splice the minigene; the amplified, processed transcript(489 base pairs) is indicated with an arrow.

Protein Electrophoresis and Western Blotting: Protein isolated fromcultures are probed using primary mouse anti-human lamin A/C monoclonalused at 1:200, MAB3211, Chemicon, followed by secondary antibody (sheepanti-mouse Fab IgG-HRP conjugate, 1:3000, Amersham) and visualized forband intensity using the Eagle Eye II Still Video System (Stratagene).

Example 11 Inhibiting Farnesylation of Progerin Prevents theCharacteristic Nuclear Blebbing of Hutchinson-Gilford Progeria Syndrome

It was hypothesized that retention of the farnesyl group causes progerinto become permanently anchored in the nuclear membrane, disruptingproper nuclear scaffolding and causing the characteristic nuclearblebbing seen in HGPS cells. Progerin's central rod domain then allowsdimerization with mature, nonfarnesylated lamin A and assembly into amultiprotein complex, resulting in dominant negative disruption of thenuclear scaffolding and underlying heterochromatin, and leading to thecharacteristic nuclear blebbing seen in HGPS (Goldman et al., Proc.Natl. Acad. Sci. USA 101:8963-8968, 2004). It was further hypothesizedthat farnesyltransferase inhibitors (FTIs) would inhibit the formationof progerin, and that decreasing the amount of this aberrant proteincould potentially improve disease status in HGPS and otherlaminopathies.

In this study, the ability of both genetic mutation and pharmacologicaltreatment to prevent the dysmorphic nuclear phenotype seen in HGPS wasexamined. The terminal CSIM sequence (SEQ ID NO: 32) in progerin wasmutated to SSIM (SEQ ID NO: 33), a sequence that cannot be farnesylated.

Material included in this example was also published as Capell et al.,Proc. Nat. Acad. Sci. 102:12879-12884, Sep. 6, 2005, which isincorporated herein by reference in its entirety.

Materials and Methods

Constructs and Mutagenesis. The pEGFP-myc-LA vector (referred to here aswild-type lamin A-CSIM) and the LAΔ50 (referred to here asprogerin-CSIM) vector were created as previously described (Goldman etal., Proc. Natl. Acad. Sci. USA 101:8963-8968, 2004) and encode greenfluorescent protein (GFP)-tagged lamin A fusion proteins. The wild-typelamin A-CSIL, progerin-CSIL, wild-type lamin A-SSIM, and progerin-SSIMmutations were created by site-directed mutagenesis using the followingoligos: for CSIL (SEQ ID NO: 13) and for SSIM (SEQ ID NO: 11)(QUIKCHANGE® II XL Site-Directed Mutagenesis Kit, Stratagene).

Cell Culture. Cell lines used were the normal human fibroblast line,AG06299, and HGPS fibroblast lines: AG06917, AG11513, and AG11498(obtained from the NIA Aging Cell Culture Repository, Coriell Institutefor Medical Research, Camden, N.J.). Fibroblasts were cultured inminimal essential medium (MEM; Invitrogen/Gibco) supplemented with 15%FBS (HiClone), 2 mM L-glutamine, penicillin (50 U/ml) and streptomycin(50 mg/ml). HeLa and HEK-293 cell lines were cultured in DMEM(Invitrogen/Gibco) supplemented with 10% fetal bovine serum andantibiotics.

Transient Transfections. Approximately 25,000 cells were plated perchamber of 4-chamber slides (Lab-Tek, Nalge Nunc International,#154526). After twenty-four hours, HeLa and HEK-293 cell lines weretransiently transfected with 0.8 μg of each construct usingLIPOFECTAMINE®2000 transfection reagent (Invitrogen) under standardconditions.

FTI Treatment. At the time of transient transfection, HeLa cells weretreated with one dose of 0, 0.5, 1.0, or 2.0 μM of the selective FTIlonafarnib [Sarasar, SCH66336, (Schering-Plough)]. HEK-293 cells weretreated with one dose of 0, 1, 2, 5, or 10 μM of the selective FTIL-744832 (Biomol). In addition, NIH-3T3 cells were treated with 5 μM ofFTI-2153, which is highly selective for FTase, or GGTI-2166, which ishighly selective for GGTase I (kind gifts of Saïd M. Sebti and Andrew D.Hamilton). These FTIs have the same mechanism of action and can be usedinterchangeably to inhibit protein farnesylation in vitro. However, onlylonafarnib is a clinical candidate. Normal and HGPS fibroblasts weretreated with one daily dose of 0, 0.5, 1.0, or 2.0 μM of lonafarnib for3 days.

GFP Localization and Fluorescence Microscopy. Forty-eight hoursfollowing transient transfection, the HeLa and HEK-293 cells werevisualized for GFP localization using an LSM510 confocal microscope(Zeiss). Following three-day treatment with lonafarnib, HGPS fibroblastswere washed two times with phosphate-buffered saline (PBS) (pH 7.2) andfixed for 10 minutes at room temperature with 1% paraformaldehyde inPBS. Following three washes with PBS, the cells were permeabilized for 5minutes at room temperature with 0.5% Triton X-100 in PBS and blockedwith 5% horse serum for 30 minutes at room temperature. Cells were thenincubated for one hour at room temperature with the primary antibodydiluted in blocking solution, a polyclonal mouse anti-human lamin A/C at1:10 (mAb3211, Chemicon). Three more washes with PBS were then followedby incubation with the secondary antibody, Alexa 488-conjugated donkeyanti-mouse IgG (Molecular Probes), for 45 minutes and three additionalPBS washes. Slides were then mounted with mounting medium containingDAPI (Vector Laboratories).

Mobility Shift Experiments. NIH-3T3 cells, grown in DMEM-H(Gibco/Invitrogen) supplemented with 10% GCS at 37° C. in 10% CO₂, weretransiently transfected with either empty vector, progerin-CSIM orprogerin-SSIM using LIPOFECTAMINE®-Plus transfection reagent(Invitrogen) according to the manufacturer's instructions. Transfectedcells were treated for 48 hours with vehicle (DMSO) or inhibitors ofprenylation (5 μM FTI-2153, 5 μM GGTI-2166 or both). Cells were lyseddirectly in 2× Laemmli loading buffer and total cell lysates wereresolved on 8% SDS PAGE. Proteins were transferred to Immobilon PVDF(Millipore), blotted with anti-GFP mAb (clone B34, Covance) andvisualized using SUPERSIGNAL™ enhanced chemiluminescence (Pierce).

Morphometric Analysis. To examine the overall percentage of blebbedcells in cells with different amounts and lengths of FTI treatment, 200nuclei were classified as either blebbed, if they contained two or morelobulations, or not blebbed, as previously described (Goldman et al.,Proc. Natl. Acad. Sci. USA 101:8963-8968, 2004). These classificationswere done by three independent observers, all blinded to the specificsof the cells being examined, followed by averaging three independentdata sets. Armitage's trend test (Armitage, Biometrics 11:375-386, 1955)was used to test for a dose-dependent response for each cell type.

Results

While both B-type lamins and lamin A are farnesylated andcarboxymethylated, unique to lamin A is a second cleavage step thatoccurs inside the cell nucleus causing the removal of an additional 15C-terminal amino acids from the mature protein, including thefarnesylated cysteine. This final cleavage step, and the resulting lossof the farnesyl anchor, is believed to release prelamin A from thenuclear membrane and allow it to be inserted into the nuclear lamina. InHGPS, although preprogerin can be farnesylated, its internal deletion ofamino acids 606-656 removes the endoprotease recognition site necessaryfor executing the final cleavage step (FIG. 21).

We sought to examine the ability of CAAX box (SEQ ID NO: 31) mutants orFTIs to block the nuclear blebbing that is the cellular hallmark of theHGPS phenotype. To examine the effects of inhibiting all prenylation,missense mutations of the cysteine residue of the CSIM motifs (SEQ IDNO: 32) of wild-type lamin A and progerin were generated (designatedSSIM). These constructs, marked for visualization by an amino-terminalGFP fusion, were transfected into HeLa cells and imaged after 48 hours.Without prenylation, all of the prelamin A was relocated from its normallocation at the nuclear periphery into nucleoplasmic aggregates, andthere was no progerin-induced nuclear blebbing (FIG. 22). Observationsby confocal microscopy suggested that these nucleoplasmic aggregateswere distributed throughout the nucleoplasm. Mutating the CSIM terminalsequence (SEQ ID NO: 32) to CSIL was predicted to favorgeranylgeranylation by GGTase I rather than farnesylation by FTase (Cox& Der, Curr. Opin. Pharmacol. 2:388-393, 2002) and therefore generate anFTI-resistant form of the protein. In this case, progerin-inducednuclear blebbing persisted (FIG. 22) indicating that the persistentmodification of any prenoid group promotes the progerin phenotype.Similar results for all of these constructs were obtained with HEK-293cells and NIH-3T3 cells.

We then explored the effect of treating HeLa cells transientlytransfected with wild-type lamin A-CSIM, progerin-CSIM, progerin-SSIM,and progerin-CSIL with the clinical candidate FTI lonafarnib (Ganguly etal., Curr. Med. Chem. 8:1419-1436, 2001). Following a single dose of 0,0.5, 1.0, 2.0 μM at the time of transfection, cells were visualized 48hours later (FIGS. 23 and 24). In the wild-type lamin A-CSIM transfectedcells, cell counting demonstrated that the percentage of nuclearblebbing was unchanged despite treatment, remaining at approximately 5%,which is the typical blebbed percentage for normal wild-type cells(Goldman et al., Proc. Natl. Acad. Sci. USA 101:8963-8968, 2004). Incontrast, those cells transfected with the progerin-CSIM constructsshowed a dramatic dose-dependent response (p<0.0001), with the nucleiapproaching normal blebbing percentages with a single dose of 2.0 μM oflonafarnib. The progerin-CSIL mutant, predicted to begeranylgeranylated, was completely resistant to treatment with the FTI,evidence that it is inhibition of the farnesylation of progerin, and notof any other endogenous farnesylated protein, that is responsible forthe improvement in nuclear phenotype seen with FTI treatment. Asexpected, there was no difference seen with FTI treatment of theprogerin-SSIM mutant, as prenylation had already been completelyinhibited. Similar results for all of these constructs were obtainedwith HEK-293 and NIH-3T3 cells using the FTIs L-744832 or FTI-2153 indoses ranging from 1.0 μM to 10 μM.

Next, the effect of FTIs on nuclear blebbing was examined in cells fromindividuals with HGPS, in this case dermal fibroblasts. Culturedfibroblasts from three HGPS patients and one unaffected mother weretreated for three days with a once daily dose of lonafarnib. In order toobserve nuclear morphology at the end of treatment, nuclei were stainedwith an antibody for lamin A and C (FIGS. 25 and 26). On day three, allthree HGPS fibroblast lines demonstrated a significant dose-dependentreduction in nuclear blebbing that was unaffected by either patient ageor cell passage number (p<0.0001).

If treatment with FTIs is to be considered a viable option for HGPS, itis important to determine whether authentic progerin, like K-Ras andsome other farnesylated proteins (Zhang et al., J. Biol. Chem.272:10232-10239, 1997) could still be geranylgeranylated in the presenceof FTIs and therefore resistant to FTI treatment. To explore thispossibility, a series of experiments were performed examining themigration of progerin on a highly resolving gel when treated with eitheran FTI, a geranylgeranyltransferase inhibitor (GGTI), or a combinationof both drugs. The appearance of a shift in mobility to a slowermigrating form is a standard method of demonstrating the ability of FTIsto prevent the processing of farnesylated proteins. Treatment with GGTIalone had no effect on the mobility of progerin (FIG. 27), demonstratingthat progerin is not normally geranylgeranylated. However, GGTI didcompletely shift the mobility of progerin-CSIL whereas FTI had noeffect, confirming that this mutant was processed as expected.Importantly, progerin treated with either an FTI or with FTI+GGTImigrated identically with vehicle-treated, completely unprocessedprogerin-SSIM mutant, demonstrating that the processing of progerin canbe completely inhibited by FTI treatment alone, and that progerin is notalternatively geranylgeranylated when farnesylation is inhibited.

Discussion

Farnesylation, a post-translational modification involving the additionof a 15-carbon isoprene moiety, was first implicated as a potentialanti-cancer target when it was discovered that the oncoprotein, Ras,which has been estimated to be involved in up to 30% of all humancancers (Bos, Cancer Res. 50:1352, 1989), required farnesylation for itsfunction (Hancock et al., Cell 57:1167, 1989; Casey et al., Proc NatlAcad Sci U S A., 86:8323, 1989; Schafer et al., Science 245:379, 1989).With the subsequent purification of the FTase enzyme (Reiss et al., Cell62:81-88, 1990), a vigorous research effort in the pharmaceuticalindustry has identified and developed a number of small moleculecompounds that potently and selectively inhibit farnesyltransferase(Sebti & Der, Nat. Rev. Cancer 3:945-951, 2003). Two of these drugs(lonafarnib/SCH66336 from Schering Plough and tipifarnib/R115777 fromJohnson and Johnson) have entered Phase III trials and have been welltolerated, including in trials involving children (Doll et al., Curr.Opin. Drug Discovery Dev. 7:478-486, 2004). Lonafarnib competes withprotein substrates for binding to the farnesyltransferase enzyme (Bishopet al., J. Biol. Chem. 270:30611-30618, 1995).

Similar to Ras, the lamin A precursor is also farnesylated, withfarnesylation serving as a required step to both insert prelamin A intothe nuclear membrane as well as to allow for the two downstream cleavagesteps which complete the processing of lamin A (Beck et al., J. CellBiol. 110:1489-1499. 1990). With the knowledge that the single C to Tbase change seen in nearly all cases of HGPS created a cryptic splicesite and thus deleted the normal second endoproteolytic cleavage site inthe lamin A processing pathway, it was hypothesized that progerin wasforced to retain its farnesyl group and could not therefore dissociateitself from the nuclear membrane. With other members of the nuclearlamina also potentially becoming trapped in complexes with themislocalized progerin, a mechanistic connection between this permanentlyfarnesylated state and the striking nuclear blebbing and disruptednuclear architecture seen in HGPS cells was proposed, and thepossibility of preventing or reversing this phenotype throughfarnesyltransferase inhibitors was raised (Eriksson et al., Nature423:293-298, 2003).

It was hypothesized that FTIs would reduce the dominant negative effectof mature progerin on nuclear membrane structure. This pharmacologiceffect would not, however, be specific for progerin; FTIs would alsodecrease levels of wild-type lamin A, increase the levels ofunfarnesylated prelamin A, and no doubt affect the posttranslationalprocessing of dozens of other farnesylated proteins. Although totalcellular lamin A would be expected to decrease, it was hypothesized thatthe net effect could still be beneficial, as the abnormal splice thatleads to progerin is produced rather inefficiently by the mutant allele,and so progerin is present in much lower quantities than wild-type laminA. Furthermore, only a small amount of mature lamin A is necessary forproper nuclear envelope assembly (Lourim & Krohne, J. Cell Biol.123:501-512, 1993). In support of this idea, clinical trials using FTIsdemonstrate little toxicity, even when levels of unfarnesylated prelaminA are significantly raised (Taveras et al., Curr. Top. Med. Chem.3:1103-1114, 2003). Complete absence of lamin A, however, leads toserious consequences and disease (Sullivan et al., J. Cell Biol.147:913-920, 1999).

The possibility that progerin would be geranylgeranylated in thepresence of FTIs was also explored. This outcome, if found, might limitthe utility of this approach. In the presence of FTIs, both oncogenicK-Ras and N-Ras serve as alternative substrates for the related enzymegeranylgeranyltransferase I (GGTase I), thus remaining biologicallyactive and fully capable of signal transduction and malignanttransformation (Ganguly et al., Curr. Med. Chem. 8:1419-1436, 2001;Whyte et al., J. Biol. Chem. 272:14459-14464, 1997; Rowell et al., J.Biol. Chem. 272:14093-14097, 1997; James et al., Proc. Natl. Acad. Sci.USA 93:4454-4458, 1996). Thus, although H-Ras does not undergoalternative prenylation when FTase activity is blocked, FTIs are noteffective inhibitors of the two Ras isoforms most commonly mutationallyactivated in human tumors, compromising the effectiveness of FTIs in thetreatment of cancer.

Fortunately, data reported here confirm that such alternativeisoprenylation does occur to any detectable degree with progerin, theprocessing of which is fully inhibited by FTI alone. These resultsindicate that farnesylated progerin is responsible for the dysmorphicnuclear morphology seen in HGPS, as inhibition of farnesylation via bothgenetic mutation and pharmacological intervention prevents nuclearblebbing and redistributes the mutant progerin protein from the nuclearmembrane into nucleoplasmic aggregates.

Beyond the prevention of this nuclear phenotype in transfected cellscaused by ectopically expressed progerin, the ability of lonafarnib tocause a significant reduction in the percentage of blebbed nuclei seenin HGPS fibroblasts, even at high passage numbers, is encouragingevidence of its therapeutic potential against endogenous progerin.Perhaps the removal of farnesylated progerin, whether through geneexpression changes or through the actual mechanical stabilization of thenuclear scaffolding, would cause the cells that are most prone to damagein HGPS to be rescued. In support of this idea is recent work showingthat by simply reducing levels of farnesylated prelamin A by 50%, thenuclear blebbing and progeria-like phenotype seen in ZMPSTE24-deficientmice could be eliminated (Fong et al., Proc. Natl Acad. Sci. USA101:18111-18116, 2004).

The results provided herein support the hypothesis that it is thepermanently farnesylated state of progerin that allows it to exert itsdominant negative effects and cause the devastating effect on nuclearstructure and function. Further, it has been demonstrated herein thatFTIs are capable of reversing this nuclear phenotype. Since FTIs arecurrently under evaluation in Phase III clinical trials as anti-cancerdrugs (Sebti & Der, Nat. Rev. Cancer 3:945-951, 2003), there isconsiderable patient information on FTI activity and toxicity.Therefore, FTIs may be a feasible therapeutic approach for HGPS.

This disclosure provides evidence that a farnesyltransferase inhibitorcan reverse or inhibit cellular effects caused by the expression ofprogerin, or caused by influence of lamin A on atherosclerosis andaging. The disclosure further provides methods and compositions thatexploit this discovery in order to treat or ameliorate HGPS and otherlaminopathies, as well as cellular aging and atherosclerosis. It will beapparent that the precise details of the methods and compositionsdescribed may be varied or modified without departing from the spirit ofthe described invention. We claim all such modifications and variationsthat fall within the scope and spirit of description and the claimsbelow.

1. A method of screening for a compound useful in treating a laminopathyin a mammal, comprising: determining if a compound alters a cellulardefect selected from the group consisting of mis-localization of afarnesylated lamin, mis-localization of a non-farnesylated lamin,nuclear membrane disruption, aggregation of lamin, nuclear lobulation,nuclear blebbing, cytoskeleton disruption, early senescence, apoptosis,reduced secretion of MMP-3, or two or more thereof; and selecting atleast one compound that alters the cellular defect so that it is moreclose to normal.
 2. A method for identifying an agent useful fortreating a laminopathy, comprising: contacting the agent to a cellexpressing progerin or a progerin-like abnormal lamin A protein underconditions sufficient to allow interaction between the cell and theagent; evaluating an amount of a cellular defect in the cell; andcomparing the amount of the cellular defect in the cell contacted withthe agent to an amount of the cellular defect in a control cell nottreated with the agent, wherein a statistically significant improvementin the amount of the cellular defect in the cell contacted with theagent, as compared to the control cell not treated with the agent,identifies the agent as one that is useful for treating the laminopathy.3. The method of claim 2, wherein the cellular defect comprisesmis-localization of a farnesylated lamin, mis-localization of anon-farnesylated lamin, nuclear membrane disruption, aggregation oflamin, nuclear lobulation, nuclear blebbing, cytoskeleton disruption,early senescence, apoptosis, reduced secretion of MMP-3, or acombination of two or more thereof.
 4. A method for identifying an agentthat inhibits or reverses at least one cellular defect caused byconstitutive farnesylation of a lamin protein, comprising: contacting acell expressing progerin or a progerin-like abnormal lamin A proteinwith a test agent under conditions sufficient to allow interactionbetween the cell and the agent; and determining whether at least onecellular defect caused by constitutive farnesylation of a lamin proteinis inhibited or reversed.
 5. The method of claim 4, wherein the cellulardefect comprises mis-localization of a farnesylated lamin,mis-localization of a non-farnesylated lamin, nuclear membranedisruption, aggregation of lamin, nuclear lobulation, nuclear blebbing,cytoskeleton disruption, early senescence, apoptosis, reduced secretionof MMP-3, or a combination of two or more thereof.